Altering Gene Expression in Modified T Cells and Uses Thereof

ABSTRACT

The present invention relates to compositions and methods for generating a modified T cell with a nucleic acid capable of downregulating endogenous gene expression selected from the group consisting of TCR α chain, TCR β chain, beta-2 microglobulin and FAS further comprising a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for a surface antigen on a target cell or an electroporated nucleic acid encoding a chimeric antigen receptor (CAR). Also included are methods and pharmaceutical compositions comprising the modified T cell for adoptive therapy and treating a condition, such as an autoimmune disease.

CROSS-REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. patent applicationSer. No. 15/516,052, filed Mar. 31, 2017, allowed, which is a 35 U.S.C.§ 371 national phase application from, and claims priority to,International Patent Application No. PCT/US2015/055780, filed Oct. 15,2015, which is entitled to priority under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 62/073,651, filed Oct. 31, 2014, allof which are incorporated by reference herein in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA120409 awardedby the National Institute of Health. The government has certain rightsin the invention.

STATEMENT REGARDING SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIAEFS-WEB

The content of the electronically submitted sequence listing in ASCIItext file (Name: 046483-7051US3-Sequence-Listing; Size: 30,644 bytes;and Date of Creation: Nov. 24, 2021) is herein incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

Adoptive cell transfer (ACT) using chimeric antigen receptor (CAR)modified T cells has been shown to be a promising strategy for thetreatment of cancers (Louis et al., 2011, Blood 118:6050-6056;Kochenderfer et al., 2010, Blood 116:3875-3886 and Porter et al., 2011,N Engl J Med 365:725-733).

Integration associated safety concerns using lentiviral or retroviralvectors are a major concern for modification of cells used for ACT. Someadvances have been made to avoid on-target or off-target unwanted sideeffects, such as RNA transfection of T cells with T cell receptor (TCR)or CAR RNA electroporation (Zhao, 2006, Mol Ther 13:151-159; Mitchell etal., Smits et al., 2004, Leukemia 18:1898-1902). By minimizing dosage ofboth RNA and T cells, such methods efficiently permit the introductionof multiple genes into cells. However, the major constraint fortransient expression of CARs is the suboptimal effector activity andfunctionality of RNA transfected T cells. Multiple T cell infusionsand/or significant use of low dose chemotherapy have been used toimprove CAR function (Barrett et al., 2013, Hum Gene Ther 24(8):717-27).

Various attempts have been made to improve effector activity andfunctionality of CARs while in order to avoid the need for combinationtherapies and additional treatments. Increasing RNA during thetransfection process poses a negative impact on T cell function,especially in vivo anti-tumor activities (Barrett et al., 2011, Hum GeneTher 22:1575-1586). Alternative constructs fusing an anti-CD3 antigenantibody fragment to an anti-tumor antigen antibody fragment have alsobeen tested in clinical trials for cancer treatments (Bargou et al.,2008, Science 321:974-977; Klinger et al., 2012, Blood 119:6226-6233).Unfortunately, these constructs were severely limited in functionalitybecause of a short half-life, poor accessibility to target cell sites,and a lack of proper long term signaling function.

Clinical TCR studies have been hampered by low expression levels of thetransduced TCR, as well as mispairing of a and R chains. Because fourTCRs can potentially be expressed at the cell surface when a T celltranscribes the chains of two different TCRs (native alpha/beta,exogenous alpha/beta, and native/exogenous “mispaired” heterodimers),significant obstacles to the use of this approach are evident. Instudies performed to date, preclinical studies have clearly demonstratedthat TCR miss-pairings have the potential to induce harmful recognitionof self-antigens.

Although early TCR and CAR T cell clinical data obtained in treatingcancers has shown promising results, the risk to the patient is high,and some patients' T cells are not potent enough for effective treatmenteven after TCR or CAR redirection, forcing modification of allogeneicdonor-derived T cells. However, the endogenous αβ T-cell receptor oninfused allogeneic T cells may recognize major and minorhistocompatibility antigens in the recipient, leading tograft-versus-host-disease (GVHD). As a result, the majority of currentclinical trials using infusion of autologous CAR T cells rely on immunetolerance to prevent TCR-mediated deleterious recognition of normaltissues after adoptive cell transfer. This approach has achieved earlyclinical successes but is limited by the time and expense to manufacturepatient-specific T-cell products. Therefore a need exists for safermethods of modifying T cells, while circumventing the time and expenseto manufacture patient-specific T-cell products.

SUMMARY OF THE INVENTION

As described herein, the present invention relates to compositions andmethods for generating a modified T cell with a nucleic acid capable ofaltering gene expression of an endogenous gene selected from the groupconsisting of TCR α chain, TCR β chain, beta-2 microglobulin, a HLAmolecule, CTLA-4, PD1, and FAS and further comprising a nucleic acidencoding a modified T cell receptor (TCR) comprising affinity for asurface antigen on a target cell.

One aspect of the invention includes a modified T cell comprising anucleic acid capable of downregulating gene expression of an endogenousgene selected from the group consisting of TCR α chain, TCR β chain,beta-2 microglobulin, a HLA molecule, CTLA-4, PD1, and FAS; and anucleic acid encoding a modified T cell receptor (TCR) comprisingaffinity for a surface antigen on a target cell.

In another aspect, the invention includes a method for generating amodified T cell comprising introducing a nucleic acid capable ofdownregulating gene expression of an endogenous gene selected from thegroup consisting of TCR α chain, TCR β chain, beta-2 microglobulin, aHLA molecule, CTLA-4, PD1, and FAS into a T cell; and introducing anucleic acid encoding a modified T cell receptor (TCR) comprisingaffinity for a surface antigen on a target cell into the T cell.

In yet another aspect, the invention includes a method of treating adisease or condition associated with enhanced immunity in a subjectcomprising administering an effective amount of a pharmaceuticalcomposition comprising the modified T cell described herein to a subjectin need thereof.

In still another aspect, the invention includes a method of treating acondition in a subject, comprising administering to the subject atherapeutically effective amount of a pharmaceutical compositioncomprising the modified T cell described herein.

In another aspect, the invention includes a method for stimulating a Tcell-mediated immune response to a target cell or tissue in a subjectcomprising administering to a subject an effective amount of apharmaceutical composition comprising the modified T cell describedherein.

In yet another aspect, the invention includes a method for adoptive celltransfer therapy comprising administering an effective amount of apharmaceutical composition comprising the modified T cell describedherein to a subject in need thereof to prevent or treat an immunereaction that is adverse to the subject.

In still another aspect, the invention includes use of the modified Tcell described herein in the manufacture of a medicament for thetreatment of an immune response in a subject in need thereof.

In another aspect, the invention includes a composition comprising themodified T cell generated according to the method described herein.

In yet another aspect, the invention includes a pharmaceuticalcomposition comprising the modified T cell generated according to themethod described herein.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the nucleic acid capable of downregulatinggene expression is selected from the group consisting of an antisenseRNA, antigomer RNA, siRNA, shRNA, and a CRISPR system, such as anpAd5/F35-CRISPR vector.

In one embodiment, the modified TCR is selected from the groupconsisting of a wildtype TCR, a high affinity TCR, and a chimeric TCR.In another embodiment, the modified TCR comprises at least one disulfidebond. In yet another embodiment, the modified TCR comprises a TCR alphachain and TCR beta chain. In still another embodiment, the modified TCRcomprises a co-stimulatory signaling domain, such as a 4-1BBco-stimulatory signaling domain, at a C′ terminal of at least one of thechains. In another embodiment, the TCR beta chain comprises at least oneN-deglycosylation. In yet another embodiment, the TCR alpha chaincomprises at least one N-deglycosylation. In still another embodiment,the modified TCR comprises at least one murine constant region.

In another embodiment, the modified TCR has higher affinity for thetarget cell surface antigen than a for a wildtype TCR. In still anotherembodiment, the target cell surface antigen is selected from the groupconsisting of viral antigen, bacterial antigen, parasitic antigen, tumorcell associated antigen (TAA), disease cell associated antigen, and anyfragment thereof.

In another embodiment, modified T cell described herein furthercomprises an exogenous nucleic acid encoding a costimulatory molecule,such as CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB, 4-1BBL, PD1 and PD1L.In one embodiment, the method of generating the modified T celldescribed herein further comprises electroporating a RNA encoding aco-stimulatory molecule into the T cell. In some embodiments where thecostimulatory molecule is CD3, the CD3 comprises at least two differentCD3 chains, such as CD3 zeta and CD3 epsilon chains.

In another embodiment, the T cell is obtained from the group consistingof peripheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line.

In yet another embodiment, the method of generating the modified T cellas described herein further comprises expanding the T cell. In oneembodiment, expanding the T cell comprises electroporating the T cellwith RNA encoding a chimeric membrane protein and culturing theelectroporated T cell. In some embodiments where chimeric membraneprotein is electroporated in the T cell, the chimeric membrane proteincomprises a single chain variable fragment (scFv) against CD3 and anintracellular domain comprises a portion of an intracellular domain ofCD28 and 4-1BB. In another embodiment, expanding the T cell comprisesculturing the T cell with a factor selected from the group consisting offlt3-L, IL-1, IL-3 and c-kit ligand.

In still another embodiment, the method of generating the modified Tcell as described herein further comprising cryopreserving the T cell.In another embodiment, the method described herein further comprisesthawing the cryopreserved T cell prior to introducing the nucleic acidinto the T cell.

In one embodiment, introducing the nucleic acid is selected from thegroup consisting of transducing the expanded T cells, transfecting theexpanded T cells, and electroporating the expanded T cells.

In yet another embodiment, the method described herein further comprisesexpressing Klf4, Oct3/4 and Sox2 in the T cells to induce pluripotencyof the T cell.

In various embodiments of the above aspects or any other aspect of theinvention delineated herein, the invention includes administering themodified T cell to a subject. In one embodiment, the subject has acondition, such as an autoimmune disease. In some embodiments, theautoimmune disease is selected from the group consisting of AcquiredImmunodeficiency Syndrome (AIDS), alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner eardisease (AIED), autoimmune lymphoproliferative syndrome (ALPS),autoimmune thrombocytopenic purpura (ATP), Behcet's disease,cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease,crest syndrome, Crohn's disease, Degos' disease,dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Meniere's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo, Wegener's granulomatosis, and anycombination thereof. In another embodiment, the condition is a cancer,such as a cancer selected from the group consisting of breast cancer,prostate cancer, ovarian cancer, cervical cancer, skin cancer,pancreatic cancer, colorectal cancer, renal cancer, liver cancer, braincancer, lymphoma, leukemia, lung cancer, and any combination thereof.

In another embodiment, the method described herein further comprisesinducing lysis, such as antibody-dependent cell-mediated cytotoxicity(ADCC), of the target cell or tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of theinvention will be better understood when read in conjunction with theappended drawings. For the purpose of illustrating the invention, thereare shown in the drawings embodiments which are presently preferred. Itshould be understood, however, that the invention is not limited to theprecise arrangements and instrumentalities of the embodiments shown inthe drawings.

FIG. 1, comprising FIGS. 1A-1C, is an illustration of the CRISPR designand targeting of the TCR αβ-CD3 complex in 293T cells. FIG. 1A shows theCRISPR gRNA targeting sites within the genomic locus of TCR-α and βconstant region. Each exon is shown by a block. Black blocks representcoding regions. Grey columns represent non-coding regions. ThirteengRNAs were designed to target exon 1 of the TCR α constant region(TRAC), 10 gRNAs target a conserved sequence on exon 1 of the TCR βconstant regions 1 (TRBC1) and 2 (TRBC2), and 10 gRNAs target exon1 ofthe beta-2 microglobin gene. FIG. 1B shows a typical gRNA scaffoldsequence (SEQ ID NO: 14). gRNA PCR products were generated by overlapPCR and cloned into MSGV vector with a T7 promoter. FIG. 1C shows Sangersequencing results (SEQ ID NOs: 69-72) showing that multiple peaks existin 293T TCR TRAC and TRBC genomic PCR products after transfection ofCAS9 mRNA and gRNAs (SEQ ID NOs: 15 and 16) into the cells.

FIG. 2, comprising FIGS. 2A-2E, shows the disruption of the TCR 4-CD3complex in primary T cells. FIG. 2A is a table showing the parametersused for electroporating CAS9 mRNA and gRNA into primary T cells withBTX830. 360V 1 ms with 2 mm cuvettes yielded the best mean fluorescentintensity (MFI) and efficiency for electroporating day 3 beadsstimulated primary T cells. FIG. 2B is a panel of graphs showing T cellsincubated at 32° C. 5% CO₂ having a much higher MFI than normal 37° C.5% CO₂ condition. FIG. 2C is a schematic illustration of the CRISPRsystem transferred into primary T cells. CAS9 mRNA and gRNA wereelectro-transferred into T cells three days after bead stimulation ofprimary T cells. T cells were then cultured with 100 IU/mL of IL-2 andsome cells were incubated at 32° C. 5% CO₂ for 1 day and then foranother 7 to 9 days. CD3 expression was analyzed on days 7-9 afterelectroporation by flow cytometry. FIG. 2D is a panel of graphs showingthat the targeting efficiency at 37° C. was about 2.5 times higher thanat 32° C. FIG. 2E is a panel of graphs showing down regulation of CD3 onday 6 after electro-transfer of varying amounts and ratios of CAS9 andgRNA targeting TCRβ. CD3 expression was analyzed by staining for CD3.The representative flow data at day 6 after electroporation is shown.Quadrant represent the percentage of CD3 negative cells in T-cellpopulations.

FIG. 3, comprising FIGS. 3A-3D, shows that TCR^(neg) alpha or beta knockout in T cells can be enriched by depletion of TCR^(pos) T cells. FIG.3A is a panel of graphs showing CD3 expression before and aftermicro-bead depletion of TCR^(neg) alpha or beta knock out in T cells.Flow cytometry illustrates expression of CD3. Numbers in the lower rightquadrant represent the percentage of CD3 negative cells in T-cellpopulations. FIG. 3B is a panel of sequencing graphs (SEQ ID NOs: 73-76)showing that multiple peaks were observed in CD3^(neg) enriched T cellgenomic PCR products. TRAC-gRNA-5 and TRBC-gRNA-7 sequences are alsoshown (SEQ ID NOs: 15 and 16). FIG. 3C is a panel of graphs showing theCD4 and CD8 T cell repertoire analysis after CD3 micro-bead enrichmentin single alpha chain, beta chain, and alpha beta double knock out Tcells modified with CRISPR. Data shows the ratio of CD8 T cellpopulation was enriched by CRISPR modification, suggesting CD8 T cellmay be more easily modified than CD4 T cells. FIG. 3D shows sequencingresults (SEQ ID NOs: 19-43) of deletions and insertions introduced tothe TCR alpha and beta locus after CRISPR modification.

FIG. 4, comprising FIGS. 4A-4C, shows that multiple electro-transfers ofgRNA greatly improved the targeting efficiency of CRISPR system inprimary T cells. FIG. 4A is a panel of graphs showing that multipleelectroporations of gRNAs greatly improved the targeting efficiency.Electroporating T cells up to three times within 24 hours gave thehighest targeting efficiency, nearly 80%. In the initial experiment,only a 15% percent of TCR targeting efficiency was achieved in T cells.Sustained expression of CAS9 was observed after electro-transfer of CAS9mRNA into the T cells. A likely reason for low cleavage efficiency maybe due to rapid degradation of gRNAs. A higher CD3 negative populationwas obtained. FIG. 4B is a panel of graphs showing that capping impairsthe function of gRNA, while early introduction of gRNAs in a secondround yielded higher efficiencies. FIG. 4C is a panel of graphs showingthat multiple electro-transfers of gRNA targeting TRAC and TRBC in ND221gave a cleavage rate of approximately 64.5% and 57.5%, respectively.

FIG. 5, comprising FIGS. 5A and 5B, shows TCR^(neg) T cells could beexpanded under different stimulating conditions. FIG. 5A is a panel ofgraphs showing that TCR^(neg) T cells restored CD3 expression afterre-introduction of TCR alpha and beta chains into TCR^(neg) T cells. CD3and Vb13.1 were detected after electroporating TCR alpha and beta chaininto TCR^(neg) T cells. CD3 expression level was comparable to TCR^(pos)T cells. FIG. 5B is a panel of graphs showing fold of expansion afterdifferent conditions used to stimulate TCR^(neg) T cells. PBMC REPyielded approximately 500 fold expansion, while CD3/CD28 beads, or K562aAPC re-stimulation yielded about 25-58 fold expansion.

FIG. 6, comprising FIGS. 6A and 6B, shows TCR^(neg) T cellcharacteristics after expansion under different conditions. FIG. 6A is apanel of graphs showing TCR^(neg) T cell phenotype characteristics afterexpansion under different conditions. FIG. 6B is a panel of graphsshowing TCR^(neg) T cell phenotype characteristics after expansion underdifferent conditions.

FIG. 7, comprising FIGS. 7A-7C, shows expanded TCR^(neg) T cells withpotent anti-tumor activity after re-direction in vitro. FIG. 7A is apanel of graphs showing that TCR^(neg) T cells could be re-directed byintroduction of an anti NY-ESO 1G4 TCR in the cells. Compared with CAS9MOCK group, when re-directed by 1G4 TCR, TCR^(neg) T cells showed ahigher level of Vb13.1 expression due to less miss-pairing of exo andendogenous TCR alpha and beta chains. FIG. 7B is a panel of graphsshowing that TCR^(neg) T cells re-directed with 1G4 TCR had highde-granulation activity when cocultured with a tumor (Nalm6-ESO) cellline. FIG. 7C is a graph showing that TCR^(neg) T cells re-directed with1G4 TCR had high cytotoxicity against a tumor cell line.

FIG. 8 is a panel of illustrations showing that directed TCR^(neg) Tcells control the growth of tumor in NSG mice after re-direction.

FIG. 9, comprising FIG. 9A-9D, shows that HLA-CLASS I elimination wasobtained by disruption of beta-2 microglobin. FIG. 9A shows sequencingdata (SEQ ID NOs: 77 and 78) of CRISPRs able to disrupt the beta-2microglobin locus in HEK293 cells. B2m-gRNA-10 sequence is also shown(SEQ ID NO: 44). FIG. 9B is a panel of graphs showing that bHLA-CLASS Inegative T cell population was generated by disruption of beta-2microglobin. FIG. 9C is a panel of graphs showing that cIFNg improvedthe targeting efficiency of beta-2 microglobin in primary T cells. FIG.9D is a panel of graphs showing that HLA-CLASS I^(neg) T cells wereenriched by microbead depletion.

FIG. 10 is a panel of graphs showing simultaneous knock out of HLA-CLASSI and TCR in primary T cells. CD4 and CD8 T cells were stimulated withCD3/CD28 dynabeads. Three days after stimulation, expanded T cells wereelectroporated with CAS9 mRNA together with TCR β constant region (TRBC)and beta-2 microglobin targeting gRNAs. Both TCR expression and beta-2microglobin expression were evaluated using anti-CD3 monoclonal antibody(mAb) and anti-beta-2 microglobin mAb six days after electroporation.Numbers represent the percentage of population in each quadrant.

FIG. 11, comprising FIGS. 11A-11D, shows triple knock out of HLA-CLASS Iand TCR alpha and beta chain in primary T cells. FIG. 11A is a panel ofgraphs showing that CD4 and CD8 T cells were stimulated with CD3/CD28dynabeads. Three days after stimulation, expanded T cells wereelectroporated with CAS9 mRNA, together with TCR alpha, beta constantregion (TRAC,TRBC) and beta-2 microglobin targeting gRNAs. Both TCRexpression and HLA-CLASS I expression were evaluated using anti-CD3monoclonal antibody (mAb) and anti-beta-2 microglobin mAb six days afterelectroporation. Numbers represent the percentage of population in eachquadrant. FIG. 11B is a schematic illustrating isolation of HLA-CLASS Iand TCR alpha and beta chain triple knock out T cells. FIG. 11C is apanel of graphs showing electroporation efficiency tested by GFPexpression. FIG. 11D is a panel of graphs showing re-introduction of TCRalpha and beta chains into TCR^(neg) T cells measured by flow cytometry.About 64% of alpha negative and about 14% beta negative population wasobserved in total TCR^(neg) T cells.

FIG. 12, comprising FIGS. 12A-12D, shows knock out of FAS in 293T cells.FIG. 12A is an image showing Sanger sequencing results (SEQ ID NOs: 79and 80) of multiple peaks when FAS was knocked out in 293T cells.FAS-gRNA-7 sequence is also shown (SEQ ID NO: 45). FIG. 12B is a panelof graphs showing FACS data revealing surface expression of FAS proteinwas disrupted by CRISPRs. FIG. 12C is a panel of images showing FASprotein was replaced by GFP after homologous recombination with CRISPRs.FIG. 12D is a panel of graphs of FACS data showing the percentage ofhomologous recombinations with CRISPRs.

FIG. 13 shows knock out of FAS in primary T cells. FACS data illustratedthat surface FAS protein expression was abolished by CRISPRs.

FIG. 14, comprising FIGS. 14A and 14B, shows knock out of PD1 in 293Tand primary T cells. FIG. 14A is an image showing Sanger sequencingresults (SEQ ID NOs: 81 and 82) of multiple peaks when PD1 were targetedin 293T cells. PD1-gRNA-2 sequence is also shown (SEQ ID NO: 46). FIG.14B is a panel of graphs showing FACS data of surface expression of PD1protein disrupted by CRISPRs.

FIG. 15, comprising FIGS. 15A and 15B, shows knock out of CTLA4 in 293Tand primary cells, such as CCD1079-SK. FIG. 15A is an image showingSanger sequencing results (SEQ ID NOs: 83 and 84) of multiple peaks whenCTLA4 were targeted in 293T cells. CTLA-4-gRNA-2 sequence is also shown(SEQ ID NO: 47). FIG. 15B is an image showing sequence data afterlimiting dilution and single cell expansion. Sanger sequencing results(SEQ ID NOs: 48-60) identified the deletions and insertions at the CTLA4genomic locus.

FIG. 16 shows knock out of PPP2r2d in 293T. Sanger sequencing data (SEQID NOs: 85-87) indicated PPP2r2d was targeted in 293T cells by CRISPRs.Ppp-trans-3,4-gRNA-1 and Ppp-trans-3,4-gRNA-2 sequences are also shown(SEQ ID NOs: 61 and 62).

FIG. 17, comprising FIGS. 17A and 17B, shows generation of iPSCs fromFAS knock out T cells. FIG. 17A is a panel of images showingmorphological change during the process of reprogramming FAS^(neg) Tcells to iPSCs. Typical embryonic stem cell morphology formationindicating FAS^(neg) T cells can be induced to pluripotent state. FIG.17B is a graph showing that FAS^(neg) T cells were reprogrammed to iPSCsat an efficiency of about 5 times of the wild type counterparts. p53deficient cell lines have been reported as easier to reprogram due tothe hinderance of the apoptosis pathway. FAS knock out may facilitatethe reprogramming process by a similar mechanism.

FIG. 18, comprising FIGS. 18A and 18B, show the generation of iPSCs fromCD3^(neg) T cells. FIG. 18A is a panel of images showing ES-likemorphology formed by CD3^(neg) TCR alpha or beta chain knock out T cellsunder defined reprogramming conditions. The morphology remains constantafter several passages. FIG. 18B is a series of graphs showing thatreprogramming CD3^(neg) T cells was about 5 time more efficient than thewild type counterparts, suggesting that TCR knock-out may play a role inthe process of T cell reprogramming or affect the cell viability afterSendai virus infection.

FIG. 19 is a graph showing knockdown of endogenous T cell receptors(TCRs) with siRNA and adding a second disulfide bond andde-N-glycosylation to the beta chain.

FIG. 20, comprising FIGS. 20A and 20B, shows TCR knockout by CAS9 RNAand gRNA. Six days after electroporation, cells were analyzed for TCRexpression by assessing CD3.

FIG. 21 is an illustration showing PCR sequencing results (SEQ ID NOs:88-91) after CD3 micro-bead depletion. TRAC-gRNA-5 and TRBC-gRNA-7sequences are also shown (SEQ ID NOs: 15 and 16).

FIG. 22 is a panel of graphs showing re-expression of CD3 four hoursafter NY-ESO-1 TCR RNA electroporation.

FIG. 23, comprising FIGS. 23A-23D, is a panel of graphs showing thatknocking down endogenous TCR enhanced both transgene expression andfunction of TCR RNA electroporated T cells. FIG. 23A shows TCRexpression of T cells electroporated with TCR siRNA (solid openhistogram), control siRNA (dotted open histogram) and T cells withoutany siRNA (filled histogram). FIG. 23B shows transgene (TCR vb13.1)expression of wild type NY-ESO-1 TCR (wt) or modified TCR (SD) RNAelectroporated T cells with TCR siRNA, control siRNA, or no siRNA. FIG.23C shows NY-ESO-1 tetramer staining of wild type NY-ESO-1 TCR (wt) ormodified TCR (SD) RNA electroporated T cells with TCR siRNA, controlsiRNA, or no siRNA. FIG. 23D shows specific lysis of a HLA-A2/NY-ESO-1positive tumor line by TCR siRNA knockdown, wildtype NY-ESO-1 TCR RNAelectroporated T cells.

FIG. 24 is a graph showing fluorescence of tumor cells after injectionof T cells into a mouse model. Ten million Nalm6-CBG-ESO-GFP (clickbeetle green) tumor cells that expressed both NY-ESO-1 and GFP wereintravenously injected into NOD/SCID mice. Five days after tumorinoculation, CBR transduced and RNA electroporated T cells were injectedas indicated in the different groups and tumor cells were detected byfluorescence.

FIG. 25 is a panel of images showing fluorescence of injected tumor andhybrid TCR T cells in mouse models over time.

FIG. 26 is a panel of images showing the generation of universal CAR19 Tcells. The top of the figure is an illustration of the protocol togenerate the universal CAR19 T cells. The graph on the left shows thepercentage of CAR19 positive T cells after lentiviral-CAR19 genetransduction. The right panel of graphs shows the percentage of TCRsingle negative and TCR/HLA-A double negative T cells before and aftersorting.

FIG. 27 is a panel of graphs and a table showing fold expansion of CD19positive cells after stimulation with irradiated CD19 presenting K562cells.

FIG. 28A is a panel of graphs showing the endogenous and transgenic geneexpression of K562-CD19 expanded cells.

FIG. 28B is a panel of graphs showing that endogenous TCR expressionremained negative in TCR single negative cells, while TCR and HLA-Aexpression remained negative in TCR/HLA-A double negative T cells afterK562-CD19 stimulated expansion

FIG. 29A is a panel of graphs showing that the majority of expandeduniversal CAR19 T cells are CD45RO positive and expressed medium levelsof CD28 expression.

FIG. 29B is a panel of graphs showing that the majority of expandeduniversal CAR19 T cells retained high levels of CD62L expression and lowlevels of CCR7 expression.

FIG. 30A is a graph showing that CRISPR gene editing did not affect theanti-tumor activity of universal CAR19 T cells in vitro.

FIG. 30B is a panel of graphs showing that the TCR single and TCR/HLA-Adouble negative CAR19 T cells showed robust lytic capacity whenchallenged with Nalm6 tumor cells.

FIG. 30C is a panel of graphs showing cytokine secretion as part of thepotent anti-tumor activity of these cells.

FIG. 30D is a panel of graphs showing TCR single ablation or TCR andHLA-A double ablation in CAR19 T cells that exhibited similarproliferation kinetics after challenge with Nalm6 tumor cells.

FIG. 31 is a panel of images showing that CRISPR gene editing did notaffect the anti-tumor activity of universal CAR19 T cells in vivo. Allthe mice receiving unmanipulated T cells and mice infused withlentiviral GFP transduced wild type T cells died within 3 weeks aftertumor cell infusion. Objective tumor regression was observed for micereceiving CAR19 T cells. CRISPR edited TCR single or TCR/HLA-A doublenegative universal CAR19 T cells showed the same anti-tumor activity.

FIG. 32A is a panel of graphs showing TCR single or TCR and HLA-A doubleablation in T cells sharply reduced alloreactivity.

FIG. 32B is a panel of graphs showing elimination of HLA-A moleculeactivated NK cells with a long period of co-culture (5 days).

FIG. 32C is a graph showing that no off-target activity was observedwhen the cells were challenged by allogeneic whole blood PBMC for 24hours in an IFNr Eispot assay.

FIG. 33 is a panel of graphs showing that FAS ablation enhanced theanti-tumor activity of CAR19 T cells. FAS negative CAR19 T cells weregenerated. FAS ablation was confirmed by flow cytometry analysis. CAR19gene expression of FASneg T cells was comparable to the wild type. Evenafter incubation with Nalm6 tumor cells for a short period of 4 hours,CD107a expression was greatly enhanced in FASneg CAR19 T cells comparedthe wild type counterpart.

FIG. 34A is a graph showing that FAS ablation in CAR19 T cells enhancedCART cell survival and proliferation under in vitro antigenicconditions. FASneg CAR19 T cells expanded faster than wild type CAR19 Tcells when the cells were stimulated with high levels of CD19+K562cells.

FIG. 34B is a panel of graphs showing FASneg CAR19 T cells had reducedapoptosis levels as measured by Annexin V staining.

FIG. 35A is a graph showing that FAS ablation in CAR19 T cells enhancedCART cell function in an animal model. As had been observed in vitro,FASneg T cells showed enhanced proliferation as compared to the wildtype T cells.

FIG. 35B is a panel of images showing FASneg CAR19 group demonstratedsuperior anti-tumor activity when compared to wild type group.

FIG. 35C is a graph showing significant differences in bioluminescencedata between FASneg CAR19 group and wild type group.

FIG. 36 is a panel of graphs showing the generation of PD1 negativePSCA-CAR T cells. PD1 ablation was confirmed by flow cytometricanalysis. PD1 negative cells were enriched by microbead depletion. Wildtype or PD1 negative PSCA-CAR T cells were expanded by stimulation withirradiated PSCA antigen presenting PC3 tumor cells. PSCA-CAR positivecells were enriched after expansion.

FIG. 37 is a panel of graphs showing that PD1 ablation and CD137expression in PSCA-CAR T cells enhanced CART cell activation under invitro antigenic conditions.

FIG. 38A is a panel of images showing PD1 ablation in an in vivoPC3-PSCA-PDL1 NSG model. The PSCA-CAR T cells demonstrated enhanced CARTcell in vivo anti-tumor activity as compared to wild type group.

FIG. 38B is a graph showing the difference in tumor burden between thePD1 negative and the wild type group.

FIG. 39 is a panel of histological images showing that T cells with TCRor TCR/HLA-I ablated did not cause graft versus host disease (GVHD). Themice treated with the double or triple knock out CART cells did notdevelop any signs of GVHD. By contrast, 3 out of 4 mice from thewild-type CD19 CART group developed GVHD by day 65, which was confirmedby histological examination of different organs.

FIG. 40A is a graph showing the percent survival of animals injectedwith T cells with TCR or TCR/HLA-I ablated. Mice were sub-lethallyirradiated and injected. Four out 5 mice receiving wild type T cellsdied of GVHD during the 60 day study. PBS treated, TCR single andTCR/HLA-I double ablated T cell treated groups did not show any signs ofGVHD.

FIG. 40B is a panel of graphs showing the body weight of mice receivingwild type T cells, PBS treated, TCR single or TCR/HLA-I double-ablated Tcells.

FIG. 41A is a panel of images showing improved anti-tumor activity ofuniversal CART cells after blocking PD1 and Fas pathways withCRISPR/Cas9. Superior anti-tumor activity was detected in PD1 knock outuniversal CD19-CART cells when injected into Nalm6-PDL1 bearing mice.

FIG. 41B is a graph showing quantitative bioluminescence data of micereceiving different CRISPR/Cas9 edited T cells.

FIG. 42 is a panel of illustrations showing a one-shot system togenerate universal CART cells. As gRNAs are prone to degrade, asimplified one-shot method was developed to constitutively express gRNAstogether with CAR in a single lentiviral vector.

FIG. 43 is a panel of graphs showing efficient gene ablation with theone-shot system. Different amounts of CD3 ablation were observed afterelectro-transfer of Cas9 mRNA.

FIG. 44A is a panel of images showing the morphological changes duringthe process of reprogramming of iPSCs from Fas knock out T cells.Typical embryonic stem cell morphology formation indicating FASnegTcells can be induced to pluripotent state.

FIG. 44B is a graph showing FASneg T cells reprogrammed to iPSCs at anefficiency of about 5 times of the wild type counterparts. p53 deficientcell lines have been reported as easier to reprogram due to thehindrance of the apoptosis pathway. FAS knock out may facilitate thereprogramming process using a similar mechanism.

FIG. 45A is a panel of images showing the ES-like morphology of iPSCsfrom CD3neg TCR alpha or beta chain knock out T cells under definedreprogramming conditions. The morphology remained constant after severalpassages.

FIG. 45B is a graph showing that reprogramming of CD3neg T cells wasabout 5 times less efficient than the wild type counterparts, suggestingthat TCR knock-out may play a role in the process of T cellreprogramming or affect the cell viability after Sendai virus infection.

FIG. 45C is a panel of images showing phosphatase staining of CD3negiPSC cells.

FIG. 46 is a panel of graphs showing induction of endogenous pluripotentstem cell genes in different T-iPSC cell lines.

FIG. 47A is a panel of images showing immunostaining for Tra-1-60 andSSEA4 expression.

FIG. 47B is an image showing the confirmation of Fas knock out of T-iPSCby Sanger sequencing (SEQ ID NO: 92).

FIG. 48A is a panel of graphs showing gene ablation in naïve T cellswith a different version of Cas9. CD3 was knocked out with dCas9 andFokI-Cas9.

FIG. 48B is a panel of graphs showing that two gRNAs were needed forgene ablation of dCas9 and FokI-Cas9.

FIG. 48C is in image showing rare off-target events in gene modified Tcells with CRISPR/cas9.

FIG. 49 is a panel of images showing the strategy of introducingCRISPR/Cas9 into T cells. Schematic representation of gRNAs driven bythe T7 promoter is shown on the left. Schematic representation of thegeneration of gene-edited antigen-specific T cells using the CRISPRsystem is shown on the right. T cells were electroporated with Cas9 mRNAand gRNAs targeting a specific gene 3 days after CD3/CD28 beadstimulation and then cultured for 24 hours at 32° C. in the presence of1L2 before being returned to the normal 37° C. culture condition.Specific gene-disrupted T cells were sorted on day 8 and redirected withCAR or TCR by lentiviral transduction or mRNA electroporation genetransfer.

FIG. 50A is a panel of graphs showing CRISPR/Cas9 mediated efficient TCRdisruption in T cells. CD3 expression of CRISPR/Cas9 edited T cellscultured at 37° C. or 32° C.

FIG. 50B is a panel of graphs showing CD3 expression of CRISPR/Cas9edited T cells cultured after sequential CRISPR RNA electroporation.

FIG. 51A is a panel of graphs showing the efficient CRISPR genedisruption that occurred in T cells. CD3 expression of T cellstransferred with CRISPR using different Cas9:gRNA ratios (upper andmiddle panel) and amount of total CRISPR RNA (lower panel).

FIG. 51B is a table showing the targeting efficiency calculated by bothflow cytometry and clonal sequencing.

FIG. 52 is an image showing the amount of TCR-targeted gene disruptionmeasured by a mismatch-selective T7 surveyor nuclease assay on DNAamplified from the cells. The calculated amount of targeted genedisruption in TRAC and TRBC is shown at the bottom. Arrows indicateexpected bands.

FIG. 53A is an image of indels (in gene disruption) observed by clonalsequence analysis of PCR amplicons after CRISPR-mediated recombinationof the TCR α and β locus (SEQ ID NOs: 19-43).

FIG. 53B is an image of a diagram of the human locus encoding the TCR αand β CRISPR gRNA targeting sites within the genomic locus of the TCR αand β constant region. Each exon is shown by a block. Arrow: sensestrand gRNA targeting site, blue arrow: antisense strand gRNA targetingsite. Multiple peaks in the Sanger sequencing results (SEQ ID NOs:93-96) show the CRISPR-mediated events of NHEJ at the TRAC and TRBCgenomic loci. TRAC-gRNA-5 and TRBC-gRNA-7 sequences are also shown (SEQID NOs: 15 and 16).

FIG. 54 is a panel of graphs showing CD3 expression in purifiedTCR^(neg) cells.

FIG. 55 is a panel of graphs showing redirection of TCR/CD3^(neg) cellsvia the electrotransfer of 1G4 TCR (α and β) or CAR19 mRNA.

FIG. 56 is a graph showing TCR/CD3^(neg) cell expansion after 10 daysusing different stimulation conditions.

FIG. 57 is a panel of graphs showing that CRISPR/Cas9 editing did notimpair antitumor efficacy of primary T cells. The phenotypes ofTCR/CD3^(neg) cells after the four different expansion techniques areshown.

FIG. 58 is a panel of graphs showing the relative CD19-CAR expressionafter electrotransfer of CD19-CAR RNA into Cas9 MOCK and TCR/CD3^(neg)cells.

FIG. 59A is a panel of graphs showing that no significant functionaldifference was observed between CD19-CAR redirected Cas9 MOCK andTCR/CD3^(neg) cells as confirmed by CD107 release assay after incubationwith Nalm6 target cells. Representative data from 3 independentexperiments are shown. Bars, standard error.

FIG. 59B is a graph showing that no significant functional differencewas observed between CD19-CAR redirected Cas9 MOCK and TCR/CD3^(neg)cells as confirmed by cytotoxicity assay after incubation with Nalm6target cells. Representative data from 3 independent experiments areshown. Bars, SE=standard error.

FIG. 59C is a panel of graphs showing that no significant functionaldifference was observed between CD19-CAR redirected Cas9 MOCK andTCR/CD3^(neg) cells as confirmed by IL2 and IFNγ secretion afterincubation with the Nalm6 target cells. Representative data from 3independent experiments are shown. Bars, SE=standard error.

FIG. 59D is a panel of images of NOD/scid/γc(−/−) mice (n=12) injectedwith 1×10⁶ Nalm6 tumor cells (i.v.) the mice were randomly sorted intothree groups. Cas9 MOCK and TCR/CD3^(neg) T cells (10×10⁶) expressingthe CD19-CAR after electroporation were injected i.v. every 4 days for atotal of three injections (arrows). Mice treated with T cellselectroporated with no RNA served as controls. Images were obtained fromthe surviving animals as indicated. Imaging commenced 1 day before thestart of T cell treatment. Bars, SE=standard error, EP=electroporation;E:T=effector to tumor ratio; arrow, time point of T cell infusion; ns,not significant. ****P<0.0001, ns by two-way ANOVA plus the Bonferronipost test.

FIG. 59E is a graph showing the radiance of the fluorescent cells.

FIG. 60 is a panel of graphs showing double and triple gene ablation byCRISPR/Cas9 to generate universal effector cells. HLA-I disruption withgRNA targeting B2M.

FIG. 61 is a flow chart of the protocol to generate universal effectorcells as described herein.

FIG. 62 is a panel of graphs showing that TCR ablation abrogatednon-specific killing activity. 624mel-CBG and PC3-CBG tumor cell lineswere incubated with T cells pre-treated with or without PHA at aneffector to target ratio of 20.1 for 24 hours and cytotoxicity wascalculated based on a luciferase assay. Data are the means±SD; n=3.

FIG. 63 is a panel of graphs showing an IFNγ Elispot assay to measureallo-reactivity of TCR and TCR/HLA disruption by challenging thegene-ablated T cells with irradiated allogenic PBMCs (left panel) orco-culturing allogenic PBMCs with irradiated gene-ablated T cells.Specific spots are shown on the y axis as the spots produced in thepresence of stimulators minus the spots produced by the effectors alone.**P<0.01 by Mann-Whitney test.

FIG. 64 is a panel of graphs showing that the disruption of theendogenous TCR by CRISPR/Cas9 improved TCR-redirected T cell function.Vb13.1 and CD3 expression is shown in T cells transfected with Cas9 mRNAalone (Cas9 Mock) or CD3^(neg) T cells with disrupted endogenous TCR αalone (α KO), β alone (β KO), α and β double (α+β KO) that wereelectroporated with NY-ESO-1 TCR α (1G4 α, 2 ug), β (1G4 β, 2 ug) or α+βRNA (1G4 α+β, 2+2 ug) RNA.

FIG. 65A is a panel of graphs showing CD107a up-regulation of the TCR(1G4) α/β RNA electroporated TCR α or β single knockout or α+β doubleknockout T cells stimulated with a HLA-A2/NY-ESO-1-positive cell line(Nalm6-ESO) or the control cell line Nalm6.

FIG. 65B is a graphs showing the lytic ability of TCR α+β RNA (1G4 TCR)electroporated TCR α or β single-knockout or α+β double-knockout T cellsshown in (a) in a luciferase-based CTL assay against Nalm6-ESO.

FIG. 66 is a panel of graphs showing Vbeta and CD3 expression in TCR α+βdouble-disrupted T cells (TCR^(neg) T cells) electroporated with twodifferent NY-ESO-1 TCR RNA (1G4 TCR, 10 ug or 8F TCR, 10 ug) comparedwith control Cas9 Mock T cells.

FIG. 67A is a panel of graphs showing CD107a up-regulation in NY-ESO-1TCR (1G4 TCR or 8F TCR) RNA electroporated TCR double-knockout CD8⁺ Tcells stimulated with the HLA-A2/NY-ESO-1-positive cell lines Nalm6-ESO,624-mel or U266. Nalm6 was used as the negative control.

FIG. 67B is a panel of graphs showing cytokine production (IL-2 andTNF-α) of NY-ESO-1 TCR (1G4 TCR or 8F TCR) RNA electroporated TCRdouble-knockout T cells after stimulation with theHLA-A2/NY-ESO-1-positive cell lines Nalm6-ESO or U266; 888mel melanomacells were used as a negative control. *P<0.05, **P<0.01****P<0.0001, bytwo-way ANOVA plus the Bonferroni post test.

FIG. 68 is a panel of images showing the generation of universal CARTcells with a combination of lentiviral gene transfer and CRISPR/Cas9electroporation. A flow chart of the generation of universal CD19-CARTcells is shown. T cells were transduced with lentiviral CD19-CAR on day1 after stimulation, and Cas9 mRNA and gRNAs targeting the TCR α and TCRβ chains and B2M were electroporated in the T cells 2 days later. TheTCR and HLA-I double-negative cell population was enriched beforere-simulation for expansion.

FIG. 69 is a panel of graphs showing CD19-CAR expression ingene-modified lenti-CD19-CAR T cells expanded by CD3/CD28 beadstimulation after 1G4 TCR electroporation.

FIG. 70 is a panel of graphs showing the phenotype of CD19-CAR T cells.

FIG. 71 is a graph showing CD107a release in TCR-negative and TCR/HLA-Idouble-negative CD19-CAR T cells. Representative data from 3 independentexperiments are shown. Bars, SE=standard error.

FIG. 72 is a panel of graphs showing cytokine secretion of TCR-negativeand TCR/HLA-I double-negative CD19-CAR T cells. Representative data from3 independent experiments are shown. Bars, SE=standard error.

FIG. 73 is a graph showing tumor lytic capability of TCR-negative andTCR/HLA-I double-negative CD19-CAR T cells. Representative data from 3independent experiments are shown. Bars, SE=standard error.

FIG. 74 a panel of graphs showing CFSE-labeled CD19-CAR andnon-transduced T cells incubated with K562 and target K562-CD19 tumorcells at a ratio of 1 to 10 for 72 hours.

FIG. 75A is a graph showing BLI from mice treated with a singleinjection on day 7 expressing CD19-CAR and GFP using a lentiviralvector. ns, no difference by two-way ANOVA plus the Bonferroni posttest. Tumors were established in NSG mice (n=4 per group) by i.v.injection of 1×10⁶ Nalm6 cells. Beginning on day 7, T cells (1×10⁷)expressing lentiviral (LV) transduced CD19-CAR were infused with asingle injection. T cells expressing LV GFP protein were injected ascontrols.

FIG. 75B is a graph showing the overall survival of mice receivingLV-GFP, LV-CD19-CAR, LV-CD19-CAR-TCR/CD3^(neg) andLV-CD19-CAR-TCR/HLA-I^(neg) T cells. ns, no difference by the log-rankMantel-Cox test.

FIG. 76 is a panel of images showing that gene-modified CAR T cellsretained antitumor efficacy and did not induce GVHD. Tumors wereestablished in NSG mice (n=4 per group) by i.v. injection with 1×10⁶Nalm6 cells. Beginning on day 7, T cells (2×10⁷) expressing LV-CD19-CARwere infused with a single injection. T cells expressing LV GFP proteinwere injected as controls. Imaging commenced 1 day before T celltreatment. Organs of randomly chosen mice from different treatmentgroups were collected on day 65 and used for CD3 immunohistochemistrystaining.

FIG. 77 is a series of schematics of vectors showing the design ofpAd5F35-CRISPR targeting PD1, Fas and TCR alpha chain.

FIG. 78 is an illustration showing the design of penton modifiedpAd5F35-CRISPR with anti-CD3 ScFv, and schematic delivery ofpAd5F35-CRISPR for knock in/out chimeric antigen receptor into T cellsin vitro and in vivo.

FIG. 79A is a graph showing Sanger sequencing (SEQ ID NO: 97) of PCRproducts flanking PD1-gRNA (SEQ ID NO: 64) targeting site.Adenoviral-pAd5F35-CRISPR-PD1 virus was transduced into MD231 cells. 3days later, genomic DNA was extracted and performed PCR.

FIG. 79B shows the sequences of the targeting events in MDA231 cellsafter Adenoviral-CRISPR manipulation. PD1 PCR products were cloned intoTOPO vector and sequenced (SEQ ID NOs: 17, 18, and 65-68).

FIG. 80 is a chart showing that a decrease in gRNA use improved T cellfold expansion and only slightly decreased knockout efficiency.

FIG. 81 is a chart showing the parameters used for optimizingelectroporation conditions to obtain high CD3/B2M knockout efficiencywith improved T cell fold expansion. Compared with standardelectroporation (EP) conditions in a 2 mm cuvette (EP #10-13) or 4 mmcuvette. High CD3/B2M knockout efficiency was observed with improved Tcell fold expansion (EP #1 and 5).

FIG. 82 is a chart showing optimization of EP conditions to achievemaximum fold expansion with tolerable knockout efficiency.

FIG. 83 is a chart showing additional optimization of EP conditions toachieve maximum fold expansion with tolerable knockout efficiency.

FIG. 84 diagrams the T cell stimulation, lentiviral transduction andCRISPR electroporation procedure.

FIG. 85 is a chart showing T cell numbers (upper chart) and foldexpansion (lower chart) after the electroporation and culturingprocedure.

FIG. 86 is a panel of graphs showing the average expansion of T cells.Fold expansion of the T cells transduced with CD19 CAR alone (TD alone)or transduced with CD19 CAR and edited with CRISPR (TD/KO) (left graph).Fold expansion of the T cells on day 10 is shown in the right graph.

FIG. 87 is a panel of flow graphs showing CD3/B2M/CAR expression at day8 of expanded T cells.

FIG. 88 is a panel of graphs showing CD3/B2M expression after CD3+ Tcell depletion.

FIG. 89 is a panel of graphs showing CD3/B2M expression on day 11 inCD19 CAR TD (transduced)/CRISPR electroporated, CD3 depleted T cells;CD19 CAR TD/CRISPR electroporated T cells; and CD19 CAR TD T cells.ND463 non-transduced (NOTD) were used as a negative control.

FIG. 90 is a panel of graphs showing CD19 CAR expression on day 11 inCD19 CAR TD (transduced)/CRISPR electroporated, CD3 depleted T cells;CD19 CAR TD/CRISPR electroporated T cells; and CD19 CAR TD T cells.ND463 non-transduced (NOTD) were used as a negative control.

FIG. 91 is a panel of graphs showing CD3/B2M/CAR expression on day 11 inCD19 CAR TD (transduced)/CRISPR electroporated, CD3 depleted T cells;CD19 CAR TD/CRISPR electroporated T cells; and CD19 CAR TD T cells.ND463 non-transduced (NOTD) were used as a negative control.

FIG. 92 is a chart summarizing CD3/B2M/CAR expression in CD19 CAR TD(transduced)/CRISPR electroporated, CD3 depleted T cells; CD19 CARTD/CRISPR electroporated T cells; and CD19 CAR TD T cells.

FIG. 93 is a panel of graphs showing CD107a up-regulation in CD19 CAR TD(transduced)/CRISPR electroporated, CD3 depleted T cells; CD19 CARTD/CRISPR electroporated T cells; and CD19 CAR TD T cells.

FIG. 94 is a panel of graphs showing lytic activity of the T cells onday 11.

FIG. 95 is a panel of graphs showing cytokine production of the T cellson day 11.

FIG. 96 is a panel of graphs showing T cell expansion. No abnormal Tcell growth was observed.

DETAILED DESCRIPTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice for testing of the present invention, the preferredmaterials and methods are described herein. In describing and claimingthe present invention, the following terminology will be used.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

“About” as used herein when referring to a measurable value such as anamount, a temporal duration, and the like, is meant to encompassvariations of ±20% or ±10%, more preferably ±5%, even more preferably±1%, and still more preferably ±0.1% from the specified value, as suchvariations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that hasbeen sufficiently stimulated to induce detectable cellularproliferation. Activation can also be associated with induced cytokineproduction, and detectable effector functions. The term “activated Tcells” refers to, among other things, T cells that are undergoing celldivision.

The term “antibody,” as used herein, refers to an immunoglobulinmolecule which specifically binds with an antigen. Antibodies can beintact immunoglobulins derived from natural sources or from recombinantsources and can be immunoreactive portions of intact immunoglobulins.Antibodies are typically tetramers of immunoglobulin molecules. Theantibodies in the present invention may exist in a variety of formsincluding, for example, polyclonal antibodies, monoclonal antibodies,Fv, Fab and F(ab)₂, as well as single chain antibodies (scFv) andhumanized antibodies (Harlow et al., 1999, In: Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow etal., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor,N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883;Bird et al., 1988, Science 242:423-426).

The term “antibody fragment” refers to a portion of an intact antibodyand refers to the antigenic determining variable regions of an intactantibody. Examples of antibody fragments include, but are not limitedto, Fab, Fab′, F(ab′)₂, and Fv fragments, linear antibodies, scFvantibodies, and multispecific antibodies formed from antibody fragments.

An “antibody heavy chain,” as used herein, refers to the larger of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations.

An “antibody light chain,” as used herein, refers to the smaller of thetwo types of polypeptide chains present in all antibody molecules intheir naturally occurring conformations. α and β light chains refer tothe two major antibody light chain isotypes.

By the term “synthetic antibody” as used herein, is meant an antibodywhich is generated using recombinant DNA technology, such as, forexample, an antibody expressed by a bacteriophage as described herein.The term should also be construed to mean an antibody which has beengenerated by the synthesis of a DNA molecule encoding the antibody andwhich DNA molecule expresses an antibody protein, or an amino acidsequence specifying the antibody, wherein the DNA or amino acid sequencehas been obtained using synthetic DNA or amino acid sequence technologywhich is available and well known in the art.

The term “antigen” or “Ag” as used herein is defined as a molecule thatprovokes an immune response. This immune response may involve eitherantibody production, or the activation of specificimmunologically-competent cells, or both. The skilled artisan willunderstand that any macromolecule, including virtually all proteins orpeptides, can serve as an antigen. Furthermore, antigens can be derivedfrom recombinant or genomic DNA. A skilled artisan will understand thatany DNA, which comprises a nucleotide sequences or a partial nucleotidesequence encoding a protein that elicits an immune response thereforeencodes an “antigen” as that term is used herein. Furthermore, oneskilled in the art will understand that an antigen need not be encodedsolely by a full length nucleotide sequence of a gene. It is readilyapparent that the present invention includes, but is not limited to, theuse of partial nucleotide sequences of more than one gene and that thesenucleotide sequences are arranged in various combinations to elicit thedesired immune response. Moreover, a skilled artisan will understandthat an antigen need not be encoded by a “gene” at all. It is readilyapparent that an antigen can be generated synthesized or can be derivedfrom a biological sample. Such a biological sample can include, but isnot limited to a tissue sample, a tumor sample, a cell or a biologicalfluid.

The term “anti-tumor effect” as used herein, refers to a biologicaleffect which can be manifested by a decrease in tumor volume, a decreasein the number of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or amelioration of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by the ability of the peptides, polynucleotides,cells and antibodies of the invention in prevention of the occurrence oftumor in the first place.

The term “auto-antigen” means, in accordance with the present invention,any self-antigen which is recognized by the immune system as beingforeign. Auto-antigens comprise, but are not limited to, cellularproteins, phosphoproteins, cellular surface proteins, cellular lipids,nucleic acids, glycoproteins, including cell surface receptors.

The term “autoimmune disease” as used herein is defined as a disorderthat results from an autoimmune response. An autoimmune disease is theresult of an inappropriate and excessive response to a self-antigen.Examples of autoimmune diseases include but are not limited to,Addision's disease, alopecia areata, ankylosing spondylitis, autoimmunehepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I),dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis,Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolyticanemia, systemic lupus erythematosus, multiple sclerosis, myastheniagravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoidarthritis, sarcoidosis, scleroderma, Sjogren's syndrome,spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema,pernicious anemia, ulcerative colitis, among others.

As used herein, the term “autologous” is meant to refer to any materialderived from the same individual to which it is later to bere-introduced into the individual.

“Allogeneic” refers to a graft derived from a different animal of thesame species.

“Xenogeneic” refers to a graft derived from an animal of a differentspecies.

The term “cancer” as used herein is defined as disease characterized bythe rapid and uncontrolled growth of aberrant cells. Cancer cells canspread locally or through the bloodstream and lymphatic system to otherparts of the body. Examples of various cancers include but are notlimited to, breast cancer, prostate cancer, ovarian cancer, cervicalcancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer,liver cancer, brain cancer, lymphoma, leukemia, lung cancer and thelike. In certain embodiments, the cancer is medullary thyroid carcinoma.

The term “chimeric antigen receptor” or “CAR,” as used herein, refers toan artificial T cell receptor that is engineered to be expressed on animmune effector cell and specifically bind an antigen. CARs may be usedas a therapy with adoptive cell transfer. T cells are removed from apatient and modified so that they express the receptors specific to aparticular form of antigen. In some embodiments, the CARs have beenexpressed with specificity to a tumor associated antigen, for example.CARs may also comprise an intracellular activation domain, atransmembrane domain and an extracellular domain comprising a tumorassociated antigen binding region. In some aspects, CARs comprisefusions of single-chain variable fragments (scFv) derived monoclonalantibodies, fused to CD3-zeta transmembrane and intracellular domain.The specificity of CAR designs may be derived from ligands of receptors(e.g., peptides). In some embodiments, a CAR can target cancers byredirecting the specificity of a T cell expressing the CAR specific fortumor associated antigens.

The term “cleavage” refers to the breakage of covalent bonds, such as inthe backbone of a nucleic acid molecule. Cleavage can be initiated by avariety of methods, including, but not limited to, enzymatic or chemicalhydrolysis of a phosphodiester bond. Both single-stranded cleavage anddouble-stranded cleavage are possible. Double-stranded cleavage canoccur as a result of two distinct single-stranded cleavage events. DNAcleavage can result in the production of either blunt ends or staggeredends. In certain embodiments, fusion polypeptides may be used fortargeting cleaved double-stranded DNA.

As used herein, the term “conservative sequence modifications” isintended to refer to amino acid modifications that do not significantlyaffect or alter the binding characteristics of the antibody containingthe amino acid sequence. Such conservative modifications include aminoacid substitutions, additions and deletions. Modifications can beintroduced into an antibody of the invention by standard techniquesknown in the art, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Conservative amino acid substitutions are ones in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, oneor more amino acid residues within the CDR regions of an antibody can bereplaced with other amino acid residues from the same side chain familyand the altered antibody can be tested for the ability to bind antigensusing the functional assays described herein.

“Co-stimulatory ligand,” as the term is used herein, includes a moleculeon an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell,and the like) that specifically binds a cognate co-stimulatory moleculeon a T cell, thereby providing a signal which, in addition to theprimary signal provided by, for instance, binding of a TCR/CD3 complexwith an MHC molecule loaded with peptide, mediates a T cell response,including, but not limited to, proliferation, activation,differentiation, and the like. A co-stimulatory ligand can include, butis not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesionmolecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. A co-stimulatory ligand also encompasses, inter alia,an antibody that specifically binds with a co-stimulatory moleculepresent on a T cell, such as, but not limited to, CD27, CD28, 4-1BB,OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1(LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specificallybinds with CD83.

A “co-stimulatory molecule” refers to the cognate binding partner on a Tcell that specifically binds with a co-stimulatory ligand, therebymediating a co-stimulatory response by the T cell, such as, but notlimited to, proliferation. Co-stimulatory molecules include, but are notlimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which incombination with a primary signal, such as TCR/CD3 ligation, leads to Tcell proliferation and/or upregulation or downregulation of keymolecules.

The term “CRISPR/CAS,” “clustered regularly interspaced shortpalindromic repeats system,” or “CRISPR” refers to DNA loci containingshort repetitions of base sequences. Each repetition is followed byshort segments of spacer DNA from previous exposures to a virus.Bacteria and archaea have evolved adaptive immune defenses termedCRISPR-CRISPR-associated (Cas) systems that use short RNA to directdegradation of foreign nucleic acids. In bacteria, the CRISPR systemprovides acquired immunity against invading foreign DNA via RNA-guidedDNA cleavage.

In the type II CRISPR/Cas system, short segments of foreign DNA, termed“spacers” are integrated within the CRISPR genomic loci and transcribedand processed into short CRISPR RNA (crRNA). These crRNAs anneal totrans-activating crRNAs (tracrRNAs) and direct sequence-specificcleavage and silencing of pathogenic DNA by Cas proteins. Recent workhas shown that target recognition by the Cas9 protein requires a “seed”sequence within the crRNA and a conserved dinucleotide-containingprotospacer adjacent motif (PAM) sequence upstream of the crRNA-bindingregion.

To direct Cas9 to cleave sequences of interest, crRNA-tracrRNA fusiontranscripts, hereafter referred to as “guide RNAs” or “gRNAs” may bedesigned, from human U6 polymerase III promoter. CRISPR/CAS mediatedgenome editing and regulation, highlighted its transformative potentialfor basic science, cellular engineering and therapeutics.

The term “CRISPRi” refers to a CRISPR system for sequence specific generepression or inhibition of gene expression, such as at thetranscriptional level.

A “disease” is a state of health of an animal wherein the animal cannotmaintain homeostasis, and wherein if the disease is not ameliorated thenthe animal's health continues to deteriorate. In contrast, a “disorder”in an animal is a state of health in which the animal is able tomaintain homeostasis, but in which the animal's state of health is lessfavorable than it would be in the absence of the disorder. Leftuntreated, a disorder does not necessarily cause a further decrease inthe animal's state of health.

The term “downregulation” as used herein refers to the decrease orelimination of gene expression of one or more genes.

“Effective amount” or “therapeutically effective amount” are usedinterchangeably herein, and refer to an amount of a compound,formulation, material, or composition, as described herein effective toachieve a particular biological result or provides a therapeutic orprophylactic benefit. Such results may include, but are not limited to,anti-tumor activity as determined by any means suitable in the art.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

As used herein “endogenous” refers to any material from or producedinside an organism, cell, tissue or system.

As used herein, the term “exogenous” refers to any material introducedfrom or produced outside an organism, cell, tissue or system.

The term “expand” as used herein refers to increasing in number, as inan increase in the number of T cells. In one embodiment, the T cellsthat are expanded ex vivo increase in number relative to the numberoriginally present in the culture. In another embodiment, the T cellsthat are expanded ex vivo increase in number relative to other celltypes in the culture. The term “ex vivo,” as used herein, refers tocells that have been removed from a living organism, (e.g., a human) andpropagated outside the organism (e.g., in a culture dish, test tube, orbioreactor).

The term “expression” as used herein is defined as the transcriptionand/or translation of a particular nucleotide sequence driven by itspromoter.

“Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., Sendai viruses, lentiviruses, retroviruses,adenoviruses, and adeno-associated viruses) that incorporate therecombinant polynucleotide.

“Homologous” as used herein, refers to the subunit sequence identitybetween two polymeric molecules, e.g., between two nucleic acidmolecules, such as, two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit; e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions; e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two sequences are homologous, the two sequences are 50%homologous; if 90% of the positions (e.g., 9 of 10), are matched orhomologous, the two sequences are 90% homologous.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′)2 or other antigen-binding subsequences of antibodies)which contain minimal sequence derived from non-human immunoglobulin.For the most part, humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are found neither in the recipient antibodynor in the imported CDR or framework sequences. These modifications aremade to further refine and optimize antibody performance. In general,the humanized antibody will comprise substantially all of at least one,and typically two, variable domains, in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin andall or substantially all of the FR regions are those of a humanimmunoglobulin sequence. The humanized antibody optimally also willcomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. For further details, see Joneset al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

“Fully human” refers to an immunoglobulin, such as an antibody, wherethe whole molecule is of human origin or consists of an amino acidsequence identical to a human form of the antibody.

“Identity” as used herein refers to the subunit sequence identitybetween two polymeric molecules particularly between two amino acidmolecules, such as, between two polypeptide molecules. When two aminoacid sequences have the same residues at the same positions; e.g., if aposition in each of two polypeptide molecules is occupied by anArginine, then they are identical at that position. The identity orextent to which two amino acid sequences have the same residues at thesame positions in an alignment is often expressed as a percentage. Theidentity between two amino acid sequences is a direct function of thenumber of matching or identical positions; e.g., if half (e.g., fivepositions in a polymer ten amino acids in length) of the positions intwo sequences are identical, the two sequences are 50% identical; if 90%of the positions (e.g., 9 of 10), are matched or identical, the twoamino acids sequences are 90% identical.

The term “immunoglobulin” or “Ig,” as used herein is defined as a classof proteins, which function as antibodies. Antibodies expressed by Bcells are sometimes referred to as the BCR (B cell receptor) or antigenreceptor. The five members included in this class of proteins are IgA,IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present inbody secretions, such as saliva, tears, breast milk, gastrointestinalsecretions and mucus secretions of the respiratory and genitourinarytracts. IgG is the most common circulating antibody. IgM is the mainimmunoglobulin produced in the primary immune response in most subjects.It is the most efficient immunoglobulin in agglutination, complementfixation, and other antibody responses, and is important in defenseagainst bacteria and viruses. IgD is the immunoglobulin that has noknown antibody function, but may serve as an antigen receptor. IgE isthe immunoglobulin that mediates immediate hypersensitivity by causingrelease of mediators from mast cells and basophils upon exposure toallergen.

The term “immune response” as used herein is defined as a cellularresponse to an antigen that occurs when lymphocytes identify antigenicmolecules as foreign and induce the formation of antibodies and/oractivate lymphocytes to remove the antigen.

As used here, “induced pluripotent stem cell” or “iPS cell” refers to apluripotent stem cell that is generated from adult cells, such as Tcells. The expression of reprogramming factors, such as Klf4, Oct3/4 andSox2, in adult cells convert the cells into pluripotent cells capable ofpropagation and differentiation into multiple cell types.

As used herein, an “instructional material” includes a publication, arecording, a diagram, or any other medium of expression which can beused to communicate the usefulness of the compositions and methods ofthe invention. The instructional material of the kit of the inventionmay, for example, be affixed to a container which contains the nucleicacid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

“Isolated” means altered or removed from the natural state. For example,a nucleic acid or a peptide naturally present in a living animal is not“isolated,” but the same nucleic acid or peptide partially or completelyseparated from the coexisting materials of its natural state is“isolated.” An isolated nucleic acid or protein can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a host cell.

The term “knockdown” as used herein refers to a decrease in geneexpression of one or more genes.

The term “knockout” as used herein refers to the ablation of geneexpression of one or more genes.

A “lentivirus” as used herein refers to a genus of the Retroviridaefamily. Lentiviruses are unique among the retroviruses in being able toinfect non-dividing cells; they can deliver a significant amount ofgenetic information into the DNA of the host cell, so they are one ofthe most efficient methods of a gene delivery vector. HIV, SIV, and FIVare all examples of lentiviruses. Vectors derived from lentivirusesoffer the means to achieve significant levels of gene transfer in vivo.

By the term “modified” as used herein, is meant a changed state orstructure of a molecule or cell of the invention. Molecules may bemodified in many ways, including chemically, structurally, andfunctionally. Cells may be modified through the introduction of nucleicacids.

By the term “modulating,” as used herein, is meant mediating adetectable increase or decrease in the level of a response in a subjectcompared with the level of a response in the subject in the absence of atreatment or compound, and/or compared with the level of a response inan otherwise identical but untreated subject. The term encompassesperturbing and/or affecting a native signal or response therebymediating a beneficial therapeutic response in a subject, preferably, ahuman.

In the context of the present invention, the following abbreviations forthe commonly occurring nucleic acid bases are used. “A” refers toadenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refersto thymidine, and “U” refers to uridine.

Unless otherwise specified, a “nucleotide sequence encoding an aminoacid sequence” includes all nucleotide sequences that are degenerateversions of each other and that encode the same amino acid sequence. Thephrase nucleotide sequence that encodes a protein or an RNA may alsoinclude introns to the extent that the nucleotide sequence encoding theprotein may in some version contain an intron(s).

The term “operably linked” refers to functional linkage between aregulatory sequence and a heterologous nucleic acid sequence resultingin expression of the latter. For example, a first nucleic acid sequenceis operably linked with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter is operablylinked to a coding sequence if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

The term “overexpressed” tumor antigen or “overexpression” of a tumorantigen is intended to indicate an abnormal level of expression of atumor antigen in a cell from a disease area like a solid tumor within aspecific tissue or organ of the patient relative to the level ofexpression in a normal cell from that tissue or organ. Patients havingsolid tumors or a hematological malignancy characterized byoverexpression of the tumor antigen can be determined by standard assaysknown in the art.

“Parenteral” administration of an immunogenic composition includes,e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), orintrasternal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain ofnucleotides. Furthermore, nucleic acids are polymers of nucleotides.Thus, nucleic acids and polynucleotides as used herein areinterchangeable. One skilled in the art has the general knowledge thatnucleic acids are polynucleotides, which can be hydrolyzed into themonomeric “nucleotides.” The monomeric nucleotides can be hydrolyzedinto nucleosides. As used herein polynucleotides include, but are notlimited to, all nucleic acid sequences which are obtained by any meansavailable in the art, including, without limitation, recombinant means,i.e., the cloning of nucleic acid sequences from a recombinant libraryor a cell genome, using ordinary cloning technology and PCR™, and thelike, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” areused interchangeably, and refer to a compound comprised of amino acidresidues covalently linked by peptide bonds. A protein or peptide mustcontain at least two amino acids, and no limitation is placed on themaximum number of amino acids that can comprise a protein's or peptide'ssequence. Polypeptides include any peptide or protein comprising two ormore amino acids joined to each other by peptide bonds. As used herein,the term refers to both short chains, which also commonly are referredto in the art as peptides, oligopeptides and oligomers, for example, andto longer chains, which generally are referred to in the art asproteins, of which there are many types. “Polypeptides” include, forexample, biologically active fragments, substantially homologouspolypeptides, oligopeptides, homodimers, heterodimers, variants ofpolypeptides, modified polypeptides, derivatives, analogs, fusionproteins, among others. The polypeptides include natural peptides,recombinant peptides, synthetic peptides, or a combination thereof.

The term “promoter” as used herein is defined as a DNA sequencerecognized by the synthetic machinery of the cell, or introducedsynthetic machinery, required to initiate the specific transcription ofa polynucleotide sequence.

As used herein, the term “promoter/regulatory sequence” means a nucleicacid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

A “constitutive” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell under most or allphysiological conditions of the cell.

An “inducible” promoter is a nucleotide sequence which, when operablylinked with a polynucleotide which encodes or specifies a gene product,causes the gene product to be produced in a cell substantially only whenan inducer which corresponds to the promoter is present in the cell.

A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide encodes or specified by a gene,causes the gene product to be produced in a cell substantially only ifthe cell is a cell of the tissue type corresponding to the promoter.

A “Sendai virus” refers to a genus of the Paramyxoviridae family. Sendaiviruses are negative, single stranded RNA viruses that do not integrateinto the host genome or alter the genetic information of the host cell.Sendai viruses have an exceptionally broad host range and are notpathogenic to humans. Used as a recombinant viral vector, Sendai virusesare capable of transient but strong gene expression.

A “signal transduction pathway” refers to the biochemical relationshipbetween a variety of signal transduction molecules that play a role inthe transmission of a signal from one portion of a cell to anotherportion of a cell. The phrase “cell surface receptor” includes moleculesand complexes of molecules capable of receiving a signal andtransmitting signal across the plasma membrane of a cell.

“Single chain antibodies” refer to antibodies formed by recombinant DNAtechniques in which immunoglobulin heavy and light chain fragments arelinked to the Fv region via an engineered span of amino acids. Variousmethods of generating single chain antibodies are known, including thosedescribed in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442;Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward etal. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041.

By the term “specifically binds,” as used herein with respect to anantibody, is meant an antibody which recognizes a specific antigen, butdoes not substantially recognize or bind other molecules in a sample.For example, an antibody that specifically binds to an antigen from onespecies may also bind to that antigen from one or more species. But,such cross-species reactivity does not itself alter the classificationof an antibody as specific. In another example, an antibody thatspecifically binds to an antigen may also bind to different allelicforms of the antigen. However, such cross reactivity does not itselfalter the classification of an antibody as specific. In some instances,the terms “specific binding” or “specifically binding,” can be used inreference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, to mean that the interaction isdependent upon the presence of a particular structure (e.g., anantigenic determinant or epitope) on the chemical species; for example,an antibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced bybinding of a stimulatory molecule (e.g., a TCR/CD3 complex) with itscognate ligand thereby mediating a signal transduction event, such as,but not limited to, signal transduction via the TCR/CD3 complex.Stimulation can mediate altered expression of certain molecules, such asdownregulation of TGF-beta, and/or reorganization of cytoskeletalstructures, and the like.

A “stimulatory molecule,” as the term is used herein, means a moleculeon a T cell that specifically binds with a cognate stimulatory ligandpresent on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when presenton an antigen presenting cell (e.g., an aAPC, a dendritic cell, aB-cell, and the like) can specifically bind with a cognate bindingpartner (referred to herein as a “stimulatory molecule”) on a T cell,thereby mediating a primary response by the T cell, including, but notlimited to, activation, initiation of an immune response, proliferation,and the like. Stimulatory ligands are well-known in the art andencompass, inter alia, an MHC Class I molecule loaded with a peptide, ananti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonistanti-CD2 antibody.

The term “subject” is intended to include living organisms in which animmune response can be elicited (e.g., mammals). A “subject” or“patient,” as used therein, may be a human or non-human mammal.Non-human mammals include, for example, livestock and pets, such asovine, bovine, porcine, canine, feline and murine mammals. Preferably,the subject is human.

As used herein, a “substantially purified” cell is a cell that isessentially free of other cell types. A substantially purified cell alsorefers to a cell which has been separated from other cell types withwhich it is normally associated in its naturally occurring state. Insome instances, a population of substantially purified cells refers to ahomogenous population of cells. In other instances, this term referssimply to cell that have been separated from the cells with which theyare naturally associated in their natural state. In some embodiments,the cells are cultured in vitro. In other embodiments, the cells are notcultured in vitro.

A “target site” or “target sequence” refers to a genomic nucleic acidsequence that defines a portion of a nucleic acid to which a bindingmolecule may specifically bind under conditions sufficient for bindingto occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complexof membrane proteins that participate in the activation of T cells inresponse to the presentation of antigen. The TCR is responsible forrecognizing antigens bound to major histocompatibility complexmolecules. TCR is composed of a heterodimer of an alpha (a) and beta (β)chain, although in some cells the TCR consists of gamma and delta (γ/δ)chains. TCRs may exist in alpha/beta and gamma/delta forms, which arestructurally similar but have distinct anatomical locations andfunctions. Each chain is composed of two extracellular domains, avariable and constant domain. In some embodiments, the TCR may bemodified on any cell comprising a TCR, including, for example, a helperT cell, a cytotoxic T cell, a memory T cell, regulatory T cell, naturalkiller T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/orprophylaxis. A therapeutic effect is obtained by suppression, remission,or eradication of a disease state.

The term “transfected” or “transformed” or “transduced” as used hereinrefers to a process by which exogenous nucleic acid is transferred orintroduced into the host cell. A “transfected” or “transformed” or“transduced” cell is one which has been transfected, transformed ortransduced with exogenous nucleic acid. The cell includes the primarysubject cell and its progeny.

To “treat” a disease as the term is used herein, means to reduce thefrequency or severity of at least one sign or symptom of a disease ordisorder experienced by a subject.

The phrase “under transcriptional control” or “operatively linked” asused herein means that the promoter is in the correct location andorientation in relation to a polynucleotide to control the initiation oftranscription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to, Sendaiviral vectors, adenoviral vectors, adeno-associated virus vectors,retroviral vectors, lentiviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention canbe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. Thisapplies regardless of the breadth of the range.

DESCRIPTION

Universal T cells that avoid graft vs host disease (GVHD) are highlydesired in the clinical setting. However, use of allogeneic T cells is arisk because of rejection by the host's immune system through therecognition of HLA-A molecules. Targeting strategies to manipulatemultiple genes are complicated and efforts have yielded low efficiencyin T cells without preventing GVHD and host vs graft reactionssimultaneously.

The FAS receptor/FAS ligand (FAS/FASL) apoptosis signaling pathwaynegatively regulates T cell function. PD1 and CTLA4 are two majorinhibitory signaling pathways in T cells. The enhanced anti-tumorimmunity that results from antibody-mediated blockade of CTLA-4, PD-1 orPD-L1 suggests the potential to improve efficiency of immunotherapies byinhibiting these pathways. The invention includes the generation ofmodified T cells where the TCR α and β chain, beta-2 microglobulin, aHLA molecule, CTLA-4, PD-1, and/or FAS are depleted as a means togenerate modified T cells with reduced immunogenicity.

The present invention includes methods and compositions for generating amodified T cell by knocking down endogenous gene expression andexpressing either a modified T cell receptor or a chimeric antigenreceptor. In some embodiments, the invention includes a method forgenerating the modified T cell. Such a modified T cell can be includedin a therapeutic composition and administered to a patient in needthereof.

Knockdown of Endogenous Gene Expression

The present invention includes downregulation of endogenous geneexpression in a T cell, such as downregulating an alpha and/or betachain of the T cell receptor (TCR), beta-2 microglobulin, CTLA-4, FAS,PD1, or a major histocompatibility complex protein such as a HLAmolecule. In one embodiment, the T cell with downregulated geneexpression has reduced immunogenicity in an allogeneic environment. Inanother embodiment, the T cell with reduced immunogenicity expresses amodified TCR or a CAR for targeted effector activity.

In one aspect, the invention includes a method for generating a modifiedT cell comprising introducing a nucleic acid into a T cell capable ofdownregulating endogenous gene expression, where the gene is selectedfrom the group consisting of TCR α chain, TCR β chain, beta-2microglobulin, a HLA molecule, CTLA-4, PD1, and FAS. Downregulatingexpression of an endogenous gene that is involved in producing an immuneresponse to a cell, such as TCR α chain, TCR β chain, beta-2microglobulin, or a HLA molecule, reduces immune-mediated rejection ofthe modified T cell. For example, downregulating expression ofendogenous TCR, MHC or beta-2 microglobulin genes removes surfacepresentation of alloantigens on the T cell that could cause rejection bythe host immune system. Also, downregulating an endogenous gene thatregulates inhibitory signaling pathways in T cells, such as CTLA-4, PD1,and/or FAS, enhances anti-tumor efficacy of the modified T cell whenexposed to an immunosuppressive microenvironment.

In one aspect, a nucleic acid capable of downregulating endogenous geneexpression is introduced, such as by electroporation, transfection, orlenti- or other viral transduction, into the T cell. In another aspect,the invention includes a modified T cell comprising an electroporatednucleic acid capable of downregulating endogenous gene expression. Inyet another aspect, a modified T cell includes an electroporated nucleicacid capable of downregulating endogenous TCR gene expression. Inanother aspect, the composition comprising the modified T cell isgenerated according to a method described herein. In yet another aspect,the invention includes a pharmaceutical composition comprising themodified T cell or a modified T cell generated according to the methoddescribed herein and a pharmaceutically acceptable carrier.

The nucleic acid capable of regulating endogenous gene expression maydownregulate the endogenous gene expression. In one embodiment, thenucleic acid capable of downregulating endogenous gene expression isselected from the group consisting of an antisense RNA, antigomer RNA,siRNA, shRNA, and a CRISPR system. Endogenous gene expression may bedownregulated, knocked-down, decreased, and/or inhibited by, forexample, an antisense RNA, antigomer RNA, siRNA, shRNA, a CRISPR system,etc.

CRISPR/Cas

The CRISPR/Cas system is a facile and efficient system for inducingtargeted genetic alterations. Target recognition by the Cas9 proteinrequires a ‘seed’ sequence within the guide RNA (gRNA) and a conserveddi-nucleotide containing protospacer adjacent motif (PAM) sequenceupstream of the gRNA-binding region. The CRISPR/CAS system can therebybe engineered to cleave virtually any DNA sequence by redesigning thegRNA in cell lines (such as 293T cells), primary cells, and CAR T cells.The CRISPR/CAS system can simultaneously target multiple genomic loci byco-expressing a single CAS9 protein with two or more gRNAs, making thissystem uniquely suited for multiple gene editing or synergisticactivation of target genes.

One example of a CRISPR/Cas system used to inhibit gene expression,CRISPRi, is described in U.S. Publication No.: 2014/0068797. CRISPRiinduces permanent gene disruption that utilizes the RNA-guided Cas9endonuclease to introduce DNA double stranded breaks which triggererror-prone repair pathways to result in frame shift mutations. Acatalytically dead Cas9 lacks endonuclease activity. When coexpressedwith a guide RNA, a DNA recognition complex is generated thatspecifically interferes with transcriptional elongation, RNA polymerasebinding, or transcription factor binding. This CRISPRi systemefficiently represses expression of targeted genes.

CRISPR/Cas gene disruption occurs when a guide nucleic acid sequencespecific for a target gene and a Cas endonuclease are introduced into acell and form a complex that enables the Cas endonuclease to introduce adouble strand break at the target gene. In one embodiment, the CRISPRsystem comprises an expression vector, such as, but not limited to, anpAd5F35-CRISPR vector. In one embodiment, a modified T cell is generatedby introducing a Cas expression vector and a guide nucleic acid sequencespecific for a gene into a T cell. In another embodiment, the Casexpression vector induces expression of Cas9 endonuclease. Otherendonucleases may also be used, including but not limited to, T7, Cas3,Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1,other nucleases known in the art, and any combination thereof.

In one embodiment, inducing the Cas expression vector comprises exposingthe T cell to an agent that activates an inducible promoter in the Casexpression vector. In such an embodiment, the Cas expression vectorincludes an inducible promoter, such as one that is inducible byexposure to an antibiotic (e.g., by tetracycline or a derivative oftetracycline, for example doxycycline). However, it should beappreciated that other inducible promoters can be used. The inducingagent can be a selective condition (e.g., exposure to an agent, forexample an antibiotic) that results in induction of the induciblepromoter. This results in expression of the Cas expression vector.

The guide nucleic acid sequence is specific for a gene and targets thatgene for Cas endonuclease-induced double strand breaks. The sequence ofthe guide nucleic acid sequence may be within a loci of the gene. In oneembodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

The guide nucleic acid sequence may be specific for any gene, such as agene that would reduce immunogenicity or reduce sensitivity to animmunosuppressive microenvironment. In one embodiment, the gene mayinclude a sequence specific for a T cell receptor (TCR) chain (such asan alpha, beta, gamma and/or delta chain), beta-2 microglobulin, FAS,PD1, a major histocompatibility complex protein (such as a HLA class Imolecule and/or HLA class II molecule), CTLA-4, or any combinationthereof.

The guide nucleic acid sequence includes a RNA sequence, a DNA sequence,a combination thereof (a RNA-DNA combination sequence), or a sequencewith synthetic nucleotides. The guide nucleic acid sequence can be asingle molecule or a double molecule. In one embodiment, the guidenucleic acid sequence comprises a single guide RNA.

T Cell Receptor

Adoptive immunotherapy with T cells harboring antigen-specific TCRs havetherapeutic potential in the treatment of cancer and certain chronicviral infections. Gene-engineering of T cells with a specific TCR hasthe advantage of redirecting the T cell to an intracellular antigen.Given that most oncogenic proteins are intracellular, development of apanel of TCRs specific to an oncogenic driver protein has great appeal.

The present invention also includes a modified T cell with downregulatedgene expression as described herein and an exogenous T cell receptor(TCR). In one aspect, the invention includes a method for generating amodified T cell comprising introducing a nucleic acid encoding amodified T cell receptor (TCR) comprising affinity for a surface antigenon a target cell into the T cell and a nucleic acid capable ofregulating endogenous gene expression selected from the group consistingof TCR α chain, TCR β chain, beta-2 microglobulin, PD1, and FAS, whereinthe T cells are capable of expressing the modified TCR.

In another aspect, the invention includes a modified T cell comprisingan exogenous nucleic acid encoding a modified T cell receptor (TCR)comprising affinity for a surface antigen on a target cell and a nucleicacid capable of downregulating endogenous gene expression selected fromthe group consisting of TCR α chain, TCR β chain, beta-2 microglobulin,PD1, and FAS, wherein the T cell expresses the modified TCR and whereinthe endogenous gene expression is downregulated in the T cell. Theinvention also includes a population of cells comprising the modified Tcell described herein.

A T cell receptor is a complex of membrane proteins that participate inthe activation of T cells in response to the presentation of antigen.Stimulation of the TCR is triggered by major histocompatibility complexmolecules (MHC) on antigen presenting cells that present antigenpeptides to the T cells and bind to the TCR complexes to induce a seriesof intracellular signaling cascades.

The TCR is generally composed of six different membrane bound chainsthat form the TCR heterodimer responsible for ligand recognition. TCRsexist in alpha/beta and gamma/delta forms, which are structurallysimilar but have distinct anatomical locations and functions. In oneembodiment, the TCR comprises a TCR alpha and TCR beta chain, such asthe nucleic acid encoding the TCR comprises a nucleic acid encoding aTCR alpha and a TCR beta chain. In another embodiment, a TCR alpha chainor a TCR beta chain or both chains comprise at least oneN-deglycosylation.

Each chain is composed of two extracellular domains, a variable andconstant domain. In one embodiment, the TCR comprises at least onemurine constant region. The constant domain is proximal to the cellmembrane, followed by a transmembrane domain and a short cytoplasmictail. In one embodiment, the modified TCR comprises a cytoplasmic domainincluding a co-stimulatory signaling domain, such as a 4-1BBco-stimulatory signaling domain. The variable domain contributes to thedetermination of the particular antigen and MHC molecule to which theTCR has binding specificity. In turn, the specificity of a T cell for aunique antigen-MHC complex resides in the particular TCR expressed bythe T cell.

Each of the constant and variable domains may include an intra-chaindisulfide bond. In one embodiment, TCR comprises at least one disulfidebond. The variable domains include the highly polymorphic loopsanalogous to the complementarity determining regions (CDRs) ofantibodies. The diversity of TCR sequences is generated via somaticrearrangement of linked variable (V), diversity (D), joining (J), andconstant genes.

Functional alpha and gamma chain polypeptides are formed by rearrangedV-J-C regions, whereas beta and delta chains consist of V-D-J-C regions.The extracellular constant domain includes a membrane proximal regionand an immunoglobulin region.

In one embodiment, the TCR includes a wildtype TCR, a high affinity TCR,and a chimeric TCR. When the TCR is modified, it may have higheraffinity for the target cell surface antigen than a wildtype TCR. Inembodiments where the TCR is a chimeric TCR, the TCR may includechimeric domains, such as the TCR comprises a co-stimulatory signalingdomain at a C′ terminal of at least one of the chains. In otherembodiment, the TCR may include a modified chain, such as a modifiedalpha or beta chain. Such modifications may include, but are not limitedto, N-deglycosylation, altered domain (such as an engineered variableregion to target a specific antigen or increase affinity), addition ofone or more disulfide bonds, entire or fragment of a chain derived froma different species, and any combination thereof.

In one embodiment, the TCR comprises specificity to a target cellantigen. The target cell surface antigen may include any type of ligandthat defines the surface of a target cell. For example, the target cellsurface antigen may be chosen to recognize a ligand that acts as a cellsurface marker on target cells associated with a particular diseasestate. Thus examples of cell surface markers that may act as ligands forthe antigen binding domain of the TCR including those associated withviral, bacterial and parasitic infections, autoimmune disease and cancercells. In one embodiment, the target cell surface antigen includes anytumor associated antigen (TAA) and viral antigen, disease cellassociated antigen, or any fragment thereof.

The target cell antigen may include any protein that can be processedand presented by major histocompability complexes. For example, thetarget antigen may be associated with a particular disease state. Thusexamples of cell markers that may act as targets of the TCR includethose associated with viral, bacterial and parasitic infections,autoimmune disease and cancer cells. In one embodiment, the targetantigen includes any of tumor associated antigens (TAA) and viralantigens, or any fragment thereof.

In one aspect, the invention includes a population of modified T cellscomprising a nucleic acid encoding a modified T cell receptor (TCR)comprising affinity for a surface antigen on a target cell and a nucleicacid capable of downregulating endogenous gene expression selected fromthe group consisting of TCR α chain, TCR β chain, beta-2 microglobulin,a HLA molecule, CTLA-4, PD1, and FAS, wherein the T cells are capable ofexpressing the modified TCR.

Techniques for engineering and expressing T cell receptors include, butare not limited to, the production of TCR heterodimers which include thenative disulphide bridge which connects the respective subunits(Garboczi, et al., (1996), Nature 384(6605): 134-41; Garboczi, et al.,(1996), J Immunol 157(12): 5403-10; Chang et al., (1994), PNAS USA 91:11408-11412; Davodeau et al., (1993), J. Biol. Chem. 268(21):15455-15460; Golden et al., (1997), J. Imm. Meth. 206: 163-169; U.S.Pat. No. 6,080,840).

Chimeric Antigen Receptor (CAR)

The present invention also includes a modified T cell with downregulatedgene expression as described herein and a CAR. Thus, the presentinvention encompasses the modified T cell comprising a CAR or a nucleicacid encoding a CAR, wherein the CAR includes an antigen binding domain,a transmembrane domain and an intracellular domain.

In one aspect, the invention includes a method of generating a modifiedT cell comprising introducing a nucleic acid capable of downregulatingendogenous gene expression selected from the group consisting of TCR αchain, TCR β chain, beta-2 microglobulin, a HLA molecule, CTLA-4, PD1,and FAS into a T cell and a nucleic acid encoding a chimeric antigenreceptor (CAR) into the T cell, wherein the CAR comprises an antigenbinding domain, a transmembrane domain and an intracellular domain of aco-stimulatory molecule.

In another aspect, the invention includes a modified T cell comprising anucleic acid capable of downregulating endogenous gene expression and anucleic acid encoding a chimeric antigen receptor (CAR), wherein thedownregulated gene expression is selected from the group consisting ofTCR α chain, TCR β chain, beta-2 microglobulin, a HLA molecule, CTLA-4,PD1, and FAS, and wherein the CAR comprises an antigen binding domain, atransmembrane domain and an intracellular domain of a co-stimulatorymolecule. In one embodiment, the modified T cell further comprises anexogenous nucleic acid encoding a modified TCR comprising affinity for asurface antigen on a target cell as described elsewhere herein. Theinvention also includes a population of cells comprising the modified Tcell described herein.

One or more domains or a fragment of a domain of the CAR may be human.In one embodiment, the present invention includes a fully human CAR. Thenucleic acid sequences coding for the desired domains can be obtainedusing recombinant methods known in the art, such as, for example byscreening libraries from cells expressing the gene, by deriving the genefrom a vector known to include the same, or by isolating directly fromcells and tissues containing the same, using standard techniques.Alternatively, the gene of interest can be produced synthetically,rather than as a cloned molecule.

Example of CARs are described in U.S. Pat. Nos. 8,911,993, 8,906,682,8,975,071, 8,916,381, 9,102,760, 9,101,584, and 9,102,761, all of whichare incorporated herein by reference in their entireties.

Antigen Binding Domain

In one embodiment, the CAR comprises an antigen binding domain thatbinds to an antigen on a target cell. Examples of cell surface markersthat may act as an antigen that binds to the antigen binding domain ofthe CAR include those associated with viral, bacterial and parasiticinfections, autoimmune disease, and cancer cells.

The choice of antigen binding domain depends upon the type and number ofantigens that are present on the surface of a target cell. For example,the antigen binding domain may be chosen to recognize an antigen thatacts as a cell surface marker on a target cell associated with aparticular disease state.

In one embodiment, the antigen binding domain binds to a tumor antigen,such as an antigen that is specific for a tumor or cancer of interest.In one embodiment, the tumor antigen of the present invention comprisesone or more antigenic cancer epitopes.

The antigen binding domain can include any domain that binds to theantigen and may include, but is not limited to, a monoclonal antibody, apolyclonal antibody, a synthetic antibody, a human antibody, a humanizedantibody, a non-human antibody, and any fragment thereof. Thus, in oneembodiment, the antigen binding domain portion comprises a mammalianantibody or a fragment thereof.

The antigen binding domain may bind one or more antigens, such as butnot limited to CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to asCD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-likemolecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptorvariant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TNF receptor familymember B cell maturation (BCMA); Tn antigen ((Tn Ag) or(GalNAcα-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptortyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6;Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule(EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunitalpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha(IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21(Testisin or PRSS21); vascular endothelial growth factor receptor 2(VEGFR2); Lewis (Y) antigen; CD24; Platelet-derived growth factorreceptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4);CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2(Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growthfactor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase;prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M);Ephrin B2; fibroblast activation protein alpha (FAP); insulin-likegrowth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX);Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2);glycoprotein 100 (gp100); oncogene fusion protein consisting ofbreakpoint cluster region (BCR) and Abelson murine leukemia viraloncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2(EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); gangliosideGM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5);high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1(TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6(CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupledreceptor class C group 5, member D (GPRC5D); chromosome X open readingframe 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK);Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion ofgloboH glycoceramide (GloboH); mammary gland differentiation antigen(NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1(HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); Gprotein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locusK 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma AlternateReading Frame Protein (TARP); Wilms tumor protein (WTi); Cancer/testisantigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la);Melanoma-associated antigen 1 (MAGE-A1); ETS translocation-variant gene6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); XAntigen Family, Member 1A (XAGE1); angiopoietin-binding cell surfacereceptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1);melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1;tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase;prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanomaantigen recognized by T cells 1 (MelanA or MART1); Rat sarcoma (Ras)mutant; human Telomerase reverse transcriptase (hTERT); sarcomatranslocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG(transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetylglucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3);Androgen receptor; Cyclin B1; v-myc avian myelocytomatosis viraloncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family MemberC (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1(CYPiB1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS orBrother of the Regulator of Imprinted Sites), Squamous Cell CarcinomaAntigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5(PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specificprotein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4);synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced GlycationEndproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2(RU2); legumain; human papilloma virus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associatedimmunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor(FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily Amember 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-typelectin domain family 12 member A (CLECl12A); bone marrow stromal cellantigen 2 (BST2); EGF-like module-containing mucin-like hormonereceptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3);Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1(IGLL1).

In some instances, it is beneficial for the antigen binding domain to bederived from the same species in which the CAR will ultimately be usedin. For example, for use in humans, it may be beneficial for the antigenbinding domain of the CAR to comprise a human antibody, humanizedantibody as described elsewhere herein, or a fragment thereof.

It is also beneficial that the antigen binding domain is operably linkedto another domain of the CAR, such as the transmembrane domain or theintracellular domain, both described elsewhere herein, for expression inthe cell. In one embodiment, a nucleic acid encoding the antigen bindingdomain is operably linked to a nucleic acid encoding a transmembranedomain and a nucleic acid encoding an intracellular domain.

Transmembrane Domain

With respect to the transmembrane domain, the CAR can be designed tocomprise a transmembrane domain that connects the antigen binding domainof the CAR to the intracellular domain. In one embodiment, thetransmembrane domain is naturally associated with one or more of thedomains in the CAR. In some instances, the transmembrane domain can beselected or modified by amino acid substitution to avoid binding of suchdomains to the transmembrane domains of the same or different surfacemembrane proteins to minimize interactions with other members of thereceptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source. Where the source is natural, the domain may be derivedfrom any membrane-bound or transmembrane protein. Transmembrane regionsof particular use in this invention may be derived from (i.e. compriseat least the transmembrane region(s) of) the alpha, beta or zeta chainof the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9,CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In someinstances, a variety of human hinges can be employed as well includingthe human Ig (immunoglobulin) hinge.

In one embodiment, the transmembrane domain may be synthetic, in whichcase it will comprise predominantly hydrophobic residues such as leucineand valine. Preferably a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.

Intracellular Domain

The intracellular domain or otherwise the cytoplasmic domain of the CARis responsible for activation of the cell in which the CAR is expressed.The term “intracellular domain” is thus meant to include any portion ofthe intracellular domain sufficient to transduce the activation signal.In one embodiment, the intracellular domain includes a domainresponsible for an effector function. The term “effector function”refers to a specialized function of a cell. Effector function of a Tcell, for example, may be cytolytic activity or helper activityincluding the secretion of cytokines.

In one embodiment, the intracellular domain of the CAR includes a domainresponsible for signal activation and/or transduction. The intracellulardomain may transmit signal activation via protein-protein interactions,biochemical changes or other response to alter the cell's metabolism,shape, gene expression, or other cellular response to activation of thechimeric intracellular signaling molecule.

Examples of an intracellular domain for use in the invention include,but are not limited to, the cytoplasmic portion of the T cell receptor(TCR) and any co-stimulatory molecule that acts in concert to initiatesignal transduction following antigen receptor engagement, as well asany derivative or variant of these elements and any synthetic sequencethat has the same functional capability. In one embodiment, theintracellular domain of the CAR comprises dual signaling domains. Thedual signaling domains may include a fragment or domain from any of themolecules described herein.

Examples of the intracellular domain include a fragment or domain fromone or more molecules or receptors including, but are not limited to,TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcRgamma, FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fcgamma RIIa, DAP10,DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83,CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127,CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha,ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD,CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1(CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRTAM, Ly9(CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A,Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162),LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, otherco-stimulatory molecules described herein, any derivative, variant, orfragment thereof, any synthetic sequence of a co-stimulatory moleculethat has the same functional capability, and any combination thereof.

In one embodiment, the intracellular domain of the CAR includes anyportion of a co-stimulatory molecule, such as at least one signalingdomain from CD3, CD27, CD28, ICOS, 4-1BB, PD-1, T cell receptor (TCR),any derivative or variant thereof, any synthetic sequence thereof thathas the same functional capability, and any combination thereof.

Between the antigen binding domain and the transmembrane domain of theCAR, or between the intracellular domain and the transmembrane domain ofthe CAR, a spacer domain may be incorporated. As used herein, the term“spacer domain” generally means any oligo- or polypeptide that functionsto link the transmembrane domain to, either the antigen binding domainor, the intracellular domain in the polypeptide chain. In oneembodiment, the spacer domain may comprise up to 300 amino acids,preferably 10 to 100 amino acids and most preferably 25 to 50 aminoacids. In another embodiment, a short oligo- or polypeptide linker,preferably between 2 and 10 amino acids in length may form the linkagebetween the transmembrane domain and the intracellular domain of theCAR. An example of a linker includes a glycine-serine doublet.

Human Antibodies

It may be preferable to use human antibodies or fragments thereof whenusing bispecific antibodies or the antigen binding domains of a CAR.Completely human antibodies are particularly desirable for therapeutictreatment of human subjects. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences,including improvements to these techniques. See, also, U.S. Pat. Nos.4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433,WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741;each of which is incorporated herein by reference in its entirety. Thebispecific antibody can also include an antibody wherein the heavy andlight chains are encoded by a nucleotide sequence derived from one ormore sources of human DNA.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. For example, it has been described that thehomozygous deletion of the antibody heavy chain joining region (JH) genein chimeric and germ-line mutant mice results in complete inhibition ofendogenous antibody production. The modified embryonic stem cells areexpanded and microinjected into blastocysts to produce chimeric mice.The chimeric mice are then bred to produce homozygous offspring whichexpress human antibodies. The transgenic mice are immunized in thenormal fashion with a selected antigen, e.g., all or a portion of apolypeptide of the invention. Antibodies directed against the target ofchoice can be obtained from the immunized, transgenic mice usingconventional hybridoma technology. The human immunoglobulin transgenesharbored by the transgenic mice rearrange during B cell differentiation,and subsequently undergo class switching and somatic mutation. Thus,using such a technique, it is possible to produce therapeutically usefulIgG, IgA, IgM and IgE antibodies, including, but not limited to, IgG1(gamma 1) and IgG3. For an overview of this technology for producinghuman antibodies, see, Lonberg and Huszar (Int. Rev. Immunol., 13:65-93(1995)). For a detailed discussion of this technology for producinghuman antibodies and human monoclonal antibodies and protocols forproducing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893,WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598,each of which is incorporated by reference herein in their entirety. Inaddition, companies such as Abgenix, Inc. (Freemont, Calif.) andGenpharm (San Jose, Calif.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above. For a specific discussion of transfer of a humangerm-line immunoglobulin gene array in germ-line mutant mice that willresult in the production of human antibodies upon antigen challenge see,e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

Human antibodies can also be derived from phage-display libraries(Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol.Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309(1996)). Phage display technology (McCafferty et al., Nature,348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of unimmunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol., 222:581-597 (1991), or Griffith et al., EMBO J.,12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905,each of which is incorporated herein by reference in its entirety.

Human antibodies may also be generated by in vitro activated B cells(see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which isincorporated herein by reference in its entirety). Human antibodies mayalso be generated in vitro using hybridoma techniques such as, but notlimited to, that described by Roder et al. (Methods Enzymol.,121:140-167 (1986)).

Humanized Antibodies

Alternatively, in some embodiments, a non-human antibody can behumanized, where specific sequences or regions of the antibody aremodified to increase similarity to an antibody naturally produced in ahuman. For instance, in the present invention, the antibody or fragmentthereof may comprise a non-human mammalian scFv. In one embodiment, theantigen binding domain portion is humanized.

A humanized antibody can be produced using a variety of techniques knownin the art, including but not limited to, CDR-grafting (see, e.g.,European Patent No. EP 239,400; International Publication No. WO91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, eachof which is incorporated herein in its entirety by reference), veneeringor resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnickaet al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.,1994, PNAS, 91:969-973, each of which is incorporated herein by itsentirety by reference), chain shuffling (see, e.g., U.S. Pat. No.5,565,332, which is incorporated herein in its entirety by reference),and techniques disclosed in, e.g., U.S. Patent Application PublicationNo. US2005/0042664, U.S. Patent Application Publication No.US2005/0048617, U.S. Pat. Nos. 6,407,213, 5,766,886, InternationalPublication No. WO 9317105, Tan et al., J. Immunol., 169:1119-25 (2002),Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods,20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84(1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto etal., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., CancerRes., 55(8):1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), andPedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which isincorporated herein in its entirety by reference. Often, frameworkresidues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well-known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature,332:323, which are incorporated herein by reference in theirentireties.)

A humanized antibody has one or more amino acid residues introduced intoit from a source which is nonhuman. These nonhuman amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Thus, humanized antibodies compriseone or more CDRs from nonhuman immunoglobulin molecules and frameworkregions from human. Humanization of antibodies is well-known in the artand can essentially be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody, i.e., CDR-grafting (EP239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567;6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents ofwhich are incorporated herein by reference herein in their entirety). Insuch humanized chimeric antibodies, substantially less than an intacthuman variable domain has been substituted by the corresponding sequencefrom a nonhuman species. In practice, humanized antibodies are typicallyhuman antibodies in which some CDR residues and possibly some framework(FR) residues are substituted by residues from analogous sites in rodentantibodies. Humanization of antibodies can also be achieved by veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, 1991, MolecularImmunology, 28(4/5):489-498; Studnicka et al., Protein Engineering,7(6):805-814 (1994); and Roguska et al., PNAS, 91:969-973 (1994)) orchain shuffling (U.S. Pat. No. 5,565,332), the contents of which areincorporated herein by reference herein in their entirety.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is to reduce antigenicity. Accordingto the so-called “best-fit” method, the sequence of the variable domainof a rodent antibody is screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol., 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987), the contents of which areincorporated herein by reference herein in their entirety). Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992);Presta et al., J. Immunol., 151:2623 (1993), the contents of which areincorporated herein by reference herein in their entirety).

Antibodies can be humanized with retention of high affinity for thetarget antigen and other favorable biological properties. According toone aspect of the invention, humanized antibodies are prepared by aprocess of analysis of the parental sequences and various conceptualhumanized products using three-dimensional models of the parental andhumanized sequences. Three-dimensional immunoglobulin models arecommonly available and are familiar to those skilled in the art.Computer programs are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind the target antigen.In this way, FR residues can be selected and combined from the recipientand import sequences so that the desired antibody characteristic, suchas increased affinity for the target antigen, is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding.

A humanized antibody retains a similar antigenic specificity as theoriginal antibody. However, using certain methods of humanization, theaffinity and/or specificity of binding of the antibody to the targetantigen may be increased using methods of “directed evolution,” asdescribed by Wu et al., J. Mol. Biol., 294:151 (1999), the contents ofwhich are incorporated herein by reference herein in their entirety.

Other Molecules

The present invention also includes the modified T cell described hereinfurther comprising a co-stimulatory molecule or a nucleic acid encodingthe co-stimulatory molecule. In one embodiment, the modified T cell ofthe invention further includes an exogenous nucleic acid encoding aco-stimulatory molecule, such that the modified T cell expresses theco-stimulatory molecule. The nucleic acid may be introduced into the Tcell by transducing the T cell, transfecting the T cell, orelectroporating the T cell. In another embodiment, the co-stimulatorymolecule is selected from CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB,4-1BBL, PD1 and PD1L. In another embodiment, the so-stimulatory moleculeincludes CD3 and comprises at least two different CD3 chains, such asCD3 zeta and CD3 epsilon chains.

In another embodiment, the modified T cell further comprises KIf4,Oct3/4, and/or Sox2 or a nucleic acid encoding Klf4, Oct3/4, and/or Sox2to induce pluripotency of the T cell. The T cell can be induced topluripotency by expressing KIf4, Oct3/4 and Sox2. Klf4, Oct3/4 and Sox2may be expressed from a nucleic acid, viral vector(s) or RNAmolecule(s). In one embodiment, a viral vector encoding for Klf4, Oct3/4and Sox2 is introduced into the T cell to induce pluripotency. Inanother embodiment, a Sendai viral vector is introduced into the T cellsto induce pluripotency, wherein the Sendai viral vector encodes Klf4,Oct3/4 and Sox2.

Introduction of Nucleic Acids

Methods of introducing nucleic acids into a cell include physical,biological and chemical methods. Physical methods for introducing apolynucleotide, such as RNA, into a host cell include calcium phosphateprecipitation, lipofection, particle bombardment, microinjection,electroporation, and the like. RNA can be introduced into target cellsusing commercially available methods which include electroporation(Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II(BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany). RNAcan also be introduced into cells using cationic liposome mediatedtransfection using lipofection, using polymer encapsulation, usingpeptide mediated transfection, or using biolistic particle deliverysystems such as “gene guns” (see, for example, Nishikawa, et al. HumGene Ther., 12(8):861-70 (2001).

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, and the like. See, forexample, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Anexemplary colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (e.g., an artificial membrane vesicle).

Lipids suitable for use can be obtained from commercial sources. Forexample, dimyristyl phosphatidylcholine (“DMPC”) can be obtained fromSigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K& K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtainedfrom Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) andother lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham,Ala.). Stock solutions of lipids in chloroform or chloroform/methanolcan be stored at about −20° C. Chloroform is used as the only solventsince it is more readily evaporated than methanol. “Liposome” is ageneric term encompassing a variety of single and multilamellar lipidvehicles formed by the generation of enclosed lipid bilayers oraggregates. Liposomes can be characterized as having vesicularstructures with a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh et al.,1991 Glycobiology 5: 505-10). However, compositions that have differentstructures in solution than the normal vesicular structure are alsoencompassed. For example, the lipids may assume a micellar structure ormerely exist as nonuniform aggregates of lipid molecules. Alsocontemplated are lipofectamine-nucleic acid complexes.

Regardless of the method used to introduce exogenous nucleic acids intoa host cell or otherwise expose a cell to the inhibitor of the presentinvention, in order to confirm the presence of the nucleic acids in thehost cell, a variety of assays may be performed. Such assays include,for example, “molecular biological” assays well known to those of skillin the art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

In one embodiment, a nucleic acid encoding a T cell receptor (TCR)comprising affinity for a surface antigen on a target cell is introducedinto the expanded T cells. The nucleic acid encoding the TCR may be thesame or separate nucleic acid that is capable of downregulatingendogenous TCR gene expression. The nucleic acid encoding the TCR may beintroduced into the T cell at the same time or sequentially with thenucleic acid capable of downregulating endogenous TCR gene expression.In one embodiment, the nucleic acid encoding the TCR is introduced priorto the nucleic acid capable of downregulating endogenous TCR geneexpression.

Moreover, the nucleic acids may be introduced by any means, such astransducing the expanded T cells, transfecting the expanded T cells, andelectroporating the expanded T cells. One nucleic acid may be introducedby one method and another nucleic acid may be introduced into the T cellby a different method.

RNA

In one embodiment, the nucleic acids introduced into the T cell are RNA.In another embodiment, the RNA is mRNA that comprises in vitrotranscribed RNA or synthetic RNA. The RNA is produced by in vitrotranscription using a polymerase chain reaction (PCR)-generatedtemplate. DNA of interest from any source can be directly converted byPCR into a template for in vitro mRNA synthesis using appropriateprimers and RNA polymerase. The source of the DNA can be, for example,genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or anyother appropriate source of DNA. The desired template for in vitrotranscription is a chimeric membrane protein. By way of example, thetemplate encodes an antibody, a fragment of an antibody or a portion ofan antibody. By way of another example, the template comprises anextracellular domain comprising a single chain variable domain of anantibody, such as anti-CD3, and an intracellular domain of aco-stimulatory molecule. In one embodiment, the template for the RNAchimeric membrane protein encodes a chimeric membrane protein comprisingan extracellular domain comprising an antigen binding domain derivedfrom an antibody to a co-stimulatory molecule, and an intracellulardomain derived from a portion of an intracellular domain of CD28 and4-1BB.

PCR can be used to generate a template for in vitro transcription ofmRNA which is then introduced into cells. Methods for performing PCR arewell known in the art. Primers for use in PCR are designed to haveregions that are substantially complementary to regions of the DNA to beused as a template for the PCR. “Substantially complementary”, as usedherein, refers to sequences of nucleotides where a majority or all ofthe bases in the primer sequence are complementary, or one or more basesare non-complementary, or mismatched. Substantially complementarysequences are able to anneal or hybridize with the intended DNA targetunder annealing conditions used for PCR. The primers can be designed tobe substantially complementary to any portion of the DNA template. Forexample, the primers can be designed to amplify the portion of a genethat is normally transcribed in cells (the open reading frame),including 5′ and 3′ UTRs. The primers can also be designed to amplify aportion of a gene that encodes a particular domain of interest. In oneembodiment, the primers are designed to amplify the coding region of ahuman cDNA, including all or portions of the 5′ and 3′ UTRs. Primersuseful for PCR are generated by synthetic methods that are well known inthe art. “Forward primers” are primers that contain a region ofnucleotides that are substantially complementary to nucleotides on theDNA template that are upstream of the DNA sequence that is to beamplified. “Upstream” is used herein to refer to a location 5, to theDNA sequence to be amplified relative to the coding strand. “Reverseprimers” are primers that contain a region of nucleotides that aresubstantially complementary to a double-stranded DNA template that aredownstream of the DNA sequence that is to be amplified. “Downstream” isused herein to refer to a location 3′ to the DNA sequence to beamplified relative to the coding strand.

Chemical structures that have the ability to promote stability and/ortranslation efficiency of the RNA may also be used. The RNA preferablyhas 5′ and 3′ UTRs. In one embodiment, the 5′ UTR is between zero and3000 nucleotides in length. The length of 5′ and 3′ UTR sequences to beadded to the coding region can be altered by different methods,including, but not limited to, designing primers for PCR that anneal todifferent regions of the UTRs. Using this approach, one of ordinaryskill in the art can modify the 5′ and 3′ UTR lengths required toachieve optimal translation efficiency following transfection of thetranscribed RNA.

The 5′ and 3′ UTRs can be the naturally occurring, endogenous 5′ and 3′UTRs for the gene of interest. Alternatively, UTR sequences that are notendogenous to the gene of interest can be added by incorporating the UTRsequences into the forward and reverse primers or by any othermodifications of the template. The use of UTR sequences that are notendogenous to the gene of interest can be useful for modifying thestability and/or translation efficiency of the RNA. For example, it isknown that AU-rich elements in 3′ UTR sequences can decrease thestability of mRNA. Therefore, 3′ UTRs can be selected or designed toincrease the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

In one embodiment, the 5′ UTR can contain the Kozak sequence of theendogenous gene. Alternatively, when a 5′ UTR that is not endogenous tothe gene of interest is being added by PCR as described above, aconsensus Kozak sequence can be redesigned by adding the 5′ UTRsequence. Kozak sequences can increase the efficiency of translation ofsome RNA transcripts, but does not appear to be required for all RNAs toenable efficient translation. The requirement for Kozak sequences formany mRNAs is known in the art. In other embodiments the 5′ UTR can bederived from an RNA virus whose RNA genome is stable in cells. In otherembodiments various nucleotide analogues can be used in the 3′ or 5′ UTRto impede exonuclease degradation of the mRNA.

To enable synthesis of RNA from a DNA template without the need for genecloning, a promoter of transcription should be attached to the DNAtemplate upstream of the sequence to be transcribed. When a sequencethat functions as a promoter for an RNA polymerase is added to the 5′end of the forward primer, the RNA polymerase promoter becomesincorporated into the PCR product upstream of the open reading framethat is to be transcribed. In one embodiment, the promoter is a T7polymerase promoter, as described elsewhere herein. Other usefulpromoters include, but are not limited to, T3 and SP6 RNA polymerasepromoters. Consensus nucleotide sequences for T7, T3 and SP6 promotersare known in the art.

In one embodiment, the mRNA has both a cap on the 5′ end and a 3′poly(A) tail which determine ribosome binding, initiation of translationand stability mRNA in the cell. On a circular DNA template, forinstance, plasmid DNA, RNA polymerase produces a long concatamericproduct which is not suitable for expression in eukaryotic cells. Thetranscription of plasmid DNA linearized at the end of the 3′ UTR resultsin normal sized mRNA which is not effective in eukaryotic transfectioneven if it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3′ endof the transcript beyond the last base of the template (Schenborn andMierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva andBerzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNAtemplate is molecular cloning. However polyA/T sequence integrated intoplasmid DNA can cause plasmid instability, which is why plasmid DNAtemplates obtained from bacterial cells are often highly contaminatedwith deletions and other aberrations. This makes cloning procedures notonly laborious and time consuming but often not reliable. That is why amethod which allows construction of DNA templates with polyA/T 3′stretch without cloning highly desirable.

The polyA/T segment of the transcriptional DNA template can be producedduring PCR by using a reverse primer containing a polyT tail, such as100T tail (size can be 50-5000 T), or after PCR by any other method,including, but not limited to, DNA ligation or in vitro recombination.Poly(A) tails also provide stability to RNAs and reduce theirdegradation. Generally, the length of a poly(A) tail positivelycorrelates with the stability of the transcribed RNA. In one embodiment,the poly(A) tail is between 100 and 5000 adenosines.

Poly(A) tails of RNAs can be further extended following in vitrotranscription with the use of a poly(A) polymerase, such as E. colipolyA polymerase (E-PAP). In one embodiment, increasing the length of apoly(A) tail from 100 nucleotides to between 300 and 400 nucleotidesresults in about a two-fold increase in the translation efficiency ofthe RNA. Additionally, the attachment of different chemical groups tothe 3′ end can increase mRNA stability. Such attachment can containmodified/artificial nucleotides, aptamers and other compounds. Forexample, ATP analogs can be incorporated into the poly(A) tail usingpoly(A) polymerase. ATP analogs can further increase the stability ofthe RNA.

5′ caps also provide stability to RNA molecules. In a preferredembodiment, RNAs produced by the methods disclosed herein include a 5′cap. The 5′ cap is provided using techniques known in the art anddescribed herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444(2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al.,Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

The RNAs produced by the methods disclosed herein can also contain aninternal ribosome entry site (IRES) sequence. The IRES sequence may beany viral, chromosomal or artificially designed sequence which initiatescap-independent ribosome binding to mRNA and facilitates the initiationof translation. Any solutes suitable for cell electroporation, which cancontain factors facilitating cellular permeability and viability such assugars, peptides, lipids, proteins, antioxidants, and surfactants can beincluded.

RNA Transfection

In some embodiments, the RNA encoding a TCR is electroporated into thecells. In one embodiment, the RNA encoding the TCR is in vitrotranscribed RNA.

The disclosed methods can be applied to the modulation of T cellactivity in basic research and therapy, in the fields of cancer, stemcells, acute and chronic infections, and autoimmune diseases, includingthe assessment of the ability of the genetically modified T cell to killa target cancer cell.

The methods also provide the ability to control the level of expressionover a wide range by changing, for example, the promoter or the amountof input RNA, making it possible to individually regulate the expressionlevel. Furthermore, the PCR-based technique of mRNA production greatlyfacilitates the design of the mRNAs with different structures andcombination of their domains.

One advantage of RNA transfection methods of the invention is that RNAtransfection is essentially transient and a vector-free. A RNA transgenecan be delivered to a lymphocyte and expressed therein following a briefin vitro cell activation, as a minimal expressing cassette without theneed for any additional viral sequences. Under these conditions,integration of the transgene into the host cell genome is unlikely.Cloning of cells is not necessary because of the efficiency oftransfection of the RNA and its ability to uniformly modify the entirelymphocyte population.

Genetic modification of T cells with in vitro-transcribed RNA (IVT-RNA)makes use of two different strategies both of which have beensuccessively tested in various animal models. Cells are transfected within vitro-transcribed RNA by means of lipofection or electroporation. Itis desirable to stabilize IVT-RNA using various modifications in orderto achieve prolonged expression of transferred IVT-RNA.

Some IVT vectors are known in the literature which are utilized in astandardized manner as template for in vitro transcription and whichhave been genetically modified in such a way that stabilized RNAtranscripts are produced. Currently protocols used in the art are basedon a plasmid vector with the following structure: a 5′ RNA polymerasepromoter enabling RNA transcription, followed by a gene of interestwhich is flanked either 3′ and/or 5′ by untranslated regions (UTR), anda 3′ polyadenyl cassette containing 50-70 A nucleotides. Prior to invitro transcription, the circular plasmid is linearized downstream ofthe polyadenyl cassette by type II restriction enzymes (recognitionsequence corresponds to cleavage site). The polyadenyl cassette thuscorresponds to the later poly(A) sequence in the transcript. As a resultof this procedure, some nucleotides remain as part of the enzymecleavage site after linearization and extend or mask the poly(A)sequence at the 3′ end. It is not clear, whether this nonphysiologicaloverhang affects the amount of protein produced intracellularly fromsuch a construct.

RNA has several advantages over more traditional plasmid or viralapproaches. Gene expression from an RNA source does not requiretranscription and the protein product is produced rapidly after thetransfection. Further, since the RNA has to only gain access to thecytoplasm, rather than the nucleus, and therefore typical transfectionmethods result in an extremely high rate of transfection. In addition,plasmid based approaches require that the promoter driving theexpression of the gene of interest be active in the cells under study.

In another aspect, the RNA construct is delivered into the cells byelectroporation. See, e.g., the formulations and methodology ofelectroporation of nucleic acid constructs into mammalian cells astaught in US 2004/0014645, US 2005/0052630A1, US 2005/0070841A1, US2004/0059285A1, US 2004/0092907A1. The various parameters includingelectric field strength required for electroporation of any known celltype are generally known in the relevant research literature as well asnumerous patents and applications in the field. See e.g., U.S. Pat. Nos.6,678,556, 7,171,264, and 7,173,116. Apparatus for therapeuticapplication of electroporation are available commercially, e.g., theMedPulser™ DNA Electroporation Therapy System (Inovio/Genetronics, SanDiego, Calif.), and are described in patents such as U.S. Pat. Nos.6,567,694; 6,516,223, 5,993,434, 6,181,964, 6,241,701, and 6,233,482;electroporation may also be used for transfection of cells in vitro asdescribed e.g. in US20070128708A1. Electroporation may also be utilizedto deliver nucleic acids into cells in vitro. Accordingly,electroporation-mediated administration into cells of nucleic acidsincluding expression constructs utilizing any of the many availabledevices and electroporation systems known to those of skill in the artpresents an exciting new means for delivering an RNA of interest to atarget cell.

In one embodiment, the method includes electroporating a RNA encoding aTCR alpha and beta chain. The TCR alpha and beta chain can be encoded onthe same or separate RNAs. When the alpha and beta are encoded byseparate RNAs, the RNA may be co-electroporated.

In another embodiment, the method may further include electroporating anucleic acid encoding a costimulatory molecule. The costimulatorymolecule nucleic acid may be co-electroporated with the TCR RNA.

Sources of T Cells

Prior to expansion, a source of T cells is obtained from a subject.Non-limiting examples of subjects include humans, dogs, cats, mice,rats, and transgenic species thereof. Preferably, the subject is ahuman. T cells can be obtained from a number of sources, includingperipheral blood mononuclear cells, bone marrow, lymph node tissue,spleen tissue, umbilical cord, and tumors. In certain embodiments, anynumber of T cell lines available in the art, may be used. In certainembodiments, T cells can be obtained from a unit of blood collected froma subject using any number of techniques known to the skilled artisan,such as Ficoll separation. In one embodiment, cells from the circulatingblood of an individual are obtained by apheresis or leukapheresis. Theapheresis product typically contains lymphocytes, including T cells,monocytes, granulocytes, B cells, other nucleated white blood cells, redblood cells, and platelets. The cells collected by apheresis may bewashed to remove the plasma fraction and to place the cells in anappropriate buffer or media, such as phosphate buffered saline (PBS) orwash solution lacks calcium and may lack magnesium or may lack many ifnot all divalent cations, for subsequent processing steps. Afterwashing, the cells may be resuspended in a variety of biocompatiblebuffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, theundesirable components of the apheresis sample may be removed and thecells directly resuspended in culture media.

In another embodiment, T cells are isolated from peripheral blood bylysing the red blood cells and depleting the monocytes, for example, bycentrifugation through a PERCOLL™ gradient. Alternatively, T cells canbe isolated from umbilical cord. In any event, a specific subpopulationof T cells can be further isolated by positive or negative selectiontechniques.

The cord blood mononuclear cells so isolated can be depleted of cellsexpressing certain antigens, including, but not limited to, CD34, CD8,CD14, CD19 and CD56. Depletion of these cells can be accomplished usingan isolated antibody, a biological sample comprising an antibody, suchas ascites, an antibody bound to a physical support, and a cell boundantibody.

Enrichment of a T cell population by negative selection can beaccomplished using a combination of antibodies directed to surfacemarkers unique to the negatively selected cells. A preferred method iscell sorting and/or selection via negative magnetic immunoadherence orflow cytometry that uses a cocktail of monoclonal antibodies directed tocell surface markers present on the cells negatively selected. Forexample, to enrich for CD4+ cells by negative selection, a monoclonalantibody cocktail typically includes antibodies to CD14, CD20, CD11b,CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negativeselection, the concentration of cells and surface (e.g., particles suchas beads) can be varied. In certain embodiments, it may be desirable tosignificantly decrease the volume in which beads and cells are mixedtogether (i.e., increase the concentration of cells), to ensure maximumcontact of cells and beads. For example, in one embodiment, aconcentration of 2 billion cells/ml is used. In one embodiment, aconcentration of 1 billion cells/ml is used. In a further embodiment,greater than 100 million cells/ml is used. In a further embodiment, aconcentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 millioncells/ml is used. In yet another embodiment, a concentration of cellsfrom 75, 80, 85, 90, 95, or 100 million cells/ml is used. In furtherembodiments, concentrations of 125 or 150 million cells/ml can be used.Using high concentrations can result in increased cell yield, cellactivation, and cell expansion.

T cells can also be frozen after the washing step, which does notrequire the monocyte-removal step. While not wishing to be bound bytheory, the freeze and subsequent thaw step provides a more uniformproduct by removing granulocytes and to some extent monocytes in thecell population. After the washing step that removes plasma andplatelets, the cells may be suspended in a freezing solution. While manyfreezing solutions and parameters are known in the art and will beuseful in this context, in a non-limiting example, one method involvesusing PBS containing 20% DMSO and 8% human serum albumin, or othersuitable cell freezing media. The cells are then frozen to −80° C. at arate of 1° per minute and stored in the vapor phase of a liquid nitrogenstorage tank. Other methods of controlled freezing may be used as wellas uncontrolled freezing immediately at −20° C. or in liquid nitrogen.

In one embodiment, the population of T cells is comprised within cellssuch as peripheral blood mononuclear cells, cord blood cells, a purifiedpopulation of T cells, and a T cell line. In another embodiment,peripheral blood mononuclear cells comprise the population of T cells.In yet another embodiment, purified T cells comprise the population of Tcells.

Chimeric Membrane Protein

Generally, T cells are expanded by contact with a surface havingattached thereto an agent that stimulates a CD3/TCR complex associatedsignal and a ligand that stimulates a co-stimulatory molecule on thesurface of the T cells. The present invention comprises a novel methodof expanding a population of T cells comprising electroporating the Tcells with RNA encoding a chimeric membrane protein and culturing theelectroporated T cells, wherein the electroporated T cells within thepopulation expand at least 10 fold. The chimeric membrane protein of theinvention comprises an extracellular and intracellular domain. Theextracellular domain comprises a target-specific binding element, suchas an antibody. In one embodiment, the chimeric membrane proteincomprises a single chain variable fragment (scFv) against CD3 and anintracellular domain derived from a portion of an intracellular domainof CD28 and 4-1BB.

Expression of the chimeric membrane protein allows interaction withother cells in the population, such as cells that express CD3, tostimulate and activate expansion of the electroporated T cells. Notwishing to be held to any particular theory, the cells that express CD3may come into contact and bind with the chimeric membrane protein thatis expressed on the surface of the electroporated T cells. At least oneT cell expressing the chimeric membrane protein interacts with anothercell expressing CD3. This interaction stimulates expansion of theelectroporated T cells.

In one embodiment, the T cells are expanded prior to downregulation ofan endogenous gene. In another embodiment, the modified T cells areexpanded.

Extracellular Domain

The present invention includes an extracellular domain comprising anantigen binding domain derived from an antibody directed against aco-stimulatory molecule. The co-stimulatory molecule can include anymolecule that co-stimulates T cells, such as, but not limited to, CD3,CD28, or a combination thereof. In one embodiment, the extracellulardomain can include an antigen binding domain derived from anti-CD3,anti-CD28, or a combination thereof. In another embodiment, theextracellular domain comprises a single chain variable fragment (scFv)against CD3.

In another embodiment, the extracellular domain can include any portionof an antibody that binds to antigen including, but not limited to, theantigen binding domain of a synthetic antibody, human antibody,humanized antibody, single domain antibody, single chain variablefragments, and fragments thereof. In some instances, it is beneficialfor the extracellular domain to be derived from the same species inwhich the chimeric membrane protein will ultimately be used in. Forexample, for use in humans, it may be beneficial for the extracellulardomain of the chimeric membrane protein to comprise a human antibody orfragment thereof. Thus, in one embodiment, the extracellular domainportion comprises a human antibody or a fragment thereof as describedelsewhere herein. Alternatively, in some embodiments, the extracellulardomain portion comprises a non-human antibody that is humanized asdescribed elsewhere herein.

Intracellular Domain

The intracellular domain or cytoplasmic domain comprises a costimulatorysignaling region. The costimulatory signaling region refers to anintracellular domain of a costimulatory molecule. Costimulatorymolecules are cell surface molecules other than antigen receptors ortheir ligands that are required for an efficient response of lymphocytesto antigen.

The cytoplasmic domain or the intracellular signaling domain of thechimeric membrane protein is responsible for activation of at least oneof effector functions of the T cell. While usually the entireintracellular signaling domain can be employed, in many cases it is notnecessary to use the entire chain. To the extent that a truncatedportion of the intracellular signaling domain is used, such truncatedportion may be used in place of the intact chain as long as ittransduces the effector function signal. The intracellular signalingdomain includes any truncated portion of the intracellular signalingdomain sufficient to transduce the effector function signal.

Nonlimiting examples of intracellular signaling domains for use in thechimeric membrane protein include any portion of the intracellulardomain of CD28, 4-1BB, T cell receptor (TCR), co-stimulatory molecules,any derivative or variant of these sequences, any synthetic sequencethat has the same functional capability, and any combination thereof. Inone embodiment, the intracellular domain comprises a portion of anintracellular domain of CD28 and 4-1BB.

Other Domains of the Chimeric Membrane Protein

Between the extracellular domain and the transmembrane domain of thechimeric membrane protein, or between the cytoplasmic domain and thetransmembrane domain of the chimeric membrane protein, there may beincorporated a spacer domain, such as an oligo- or polypeptide thatfunctions to link the transmembrane domain to, either the extracellulardomain or, the cytoplasmic domain in the polypeptide chain. The spacerdomain may comprise up to 300 amino acids, preferably 10 to 100 aminoacids and most preferably 25 to 50 amino acids.

In some embodiments, the chimeric membrane protein further comprises atransmembrane domain. In some embodiment, the chimeric membrane proteinfurther comprises a hinge domain. In one embodiment, the RNA encodingthe chimeric membrane protein further comprises a transmembrane andhinge domain, such as a CD28 transmembrane domain and a CD8-alpha hingedomain.

Expansion of T Cells

As demonstrated by the data disclosed herein, expanding the T cells bythe methods disclosed herein can be multiplied by about 10 fold, 20fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold,6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold,1,000,000 fold, 10,000,000 fold, or greater, and any and all whole orpartial integers therebetween. In one embodiment, the T cells expand inthe range of about 20 fold to about 50 fold.

Following culturing, the T cells can be incubated in cell medium in aculture apparatus for a period of time or until the cells reachconfluency or high cell density for optimal passage before passing thecells to another culture apparatus. The culturing apparatus can be ofany culture apparatus commonly used for culturing cells in vitro.Preferably, the level of confluence is 70% or greater before passing thecells to another culture apparatus. More preferably, the level ofconfluence is 90% or greater. A period of time can be any time suitablefor the culture of cells in vitro. The T cell medium may be replacedduring the culture of the T cells at any time. Preferably, the T cellmedium is replaced about every 2 to 3 days. The T cells are thenharvested from the culture apparatus whereupon the T cells can be usedimmediately or cryopreserved to be stored for use at a later time. Inone embodiment, the invention includes cryopreserving the expanded Tcells. The cryopreserved T cells are thawed prior to introducing nucleicacids into the T cell.

In another embodiment, the method comprises isolating T cells andexpanding the T cells. In another embodiment, the invention furthercomprises cryopreserving the T cells prior to expansion. In yet anotherembodiment, the cryopreserved T cells are thawed for electroporationwith the RNA encoding the chimeric membrane protein.

Another procedure for ex vivo expansion cells is described in U.S. Pat.No. 5,199,942 (incorporated herein by reference). Expansion, such asdescribed in U.S. Pat. No. 5,199,942 can be an alternative or inaddition to other methods of expansion described herein. Briefly, exvivo culture and expansion of T cells comprises the addition to thecellular growth factors, such as those described in U.S. Pat. No.5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kitligand. In one embodiment, expanding the T cells comprises culturing theT cells with a factor selected from the group consisting of flt3-L,IL-1, IL-3 and c-kit ligand.

The culturing step as described herein (contact with agents as describedherein or after electroporation) can be very short, for example lessthan 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as describedfurther herein (contact with agents as described herein) can be longer,for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refersgenerally to cells taken from a living organism and grown undercontrolled condition. A primary cell culture is a culture of cells,tissues or organs taken directly from an organism and before the firstsubculture. Cells are expanded in culture when they are placed in agrowth medium under conditions that facilitate cell growth and/ordivision, resulting in a larger population of the cells. When cells areexpanded in culture, the rate of cell proliferation is typicallymeasured by the amount of time required for the cells to double innumber, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells aresubcultured, they are referred to as having been passaged. A specificpopulation of cells, or a cell line, is sometimes referred to orcharacterized by the number of times it has been passaged. For example,a cultured cell population that has been passaged ten times may bereferred to as a P10 culture. The primary culture, i.e., the firstculture following the isolation of cells from tissue, is designated P0.Following the first subculture, the cells are described as a secondaryculture (P1 or passage 1). After the second subculture, the cells becomea tertiary culture (P2 or passage 2), and so on. It will be understoodby those of skill in the art that there may be many population doublingsduring the period of passaging; therefore the number of populationdoublings of a culture is greater than the passage number. The expansionof cells (i.e., the number of population doublings) during the periodbetween passaging depends on many factors, including but is not limitedto the seeding density, substrate, medium, and time between passaging.

In one embodiment, the cells may be cultured for several hours (about 3hours) to about 14 days or any hourly integer value in between.Conditions appropriate for T cell culture include an appropriate media(e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15,(Lonza)) that may contain factors necessary for proliferation andviability, including serum (e.g., fetal bovine or human serum),interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10,IL-12, IL-15, TGF-beta, and TNF-α. or any other additives for the growthof cells known to the skilled artisan. Other additives for the growth ofcells include, but are not limited to, surfactant, plasmanate, andreducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Mediacan include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, andX-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, andvitamins, either serum-free or supplemented with an appropriate amountof serum (or plasma) or a defined set of hormones, and/or an amount ofcytokine(s) sufficient for the growth and expansion of T cells.Antibiotics, e.g., penicillin and streptomycin, are included only inexperimental cultures, not in cultures of cells that are to be infusedinto a subject. The target cells are maintained under conditionsnecessary to support growth, for example, an appropriate temperature(e.g., 37° C.) and atmosphere (e.g., air plus 5% CO₂).

The medium used to culture the T cells may include an agent that canco-stimulate the T cells. For example, an agent that can stimulate CD3is an antibody to CD3, and an agent that can stimulate CD28 is anantibody to CD28. This is because, as demonstrated by the data disclosedherein, a cell isolated by the methods disclosed herein can be expandedapproximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold,10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater.In one embodiment, the T cells expand in the range of about 20 fold toabout 50 fold, or more by culturing the electroporated population.

In one embodiment, the method includes introducing a nucleic acidencoding a T cell receptor (TCR) comprising affinity for a surfaceantigen on a target cell into the expanded T cells, and electroporatinga RNA encoding a co-stimulatory molecule into the T cells, wherein theelectroporated T cells are capable of expressing the TCR and theco-stimulatory molecule.

In another embodiment, the method further comprises stimulating theexpanded T cells with at least one co-stimulatory molecule selected fromthe group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB,4-1BBL, PD1 and PD1L. The stimulation may include co-electroporationwith RNA encoding the co-stimulatory molecule. In such an embodiment,the expanded T cells are further electroporated or co-electroporatedwith a RNA encoding CD3. The CD3 includes comprises at least twodifferent CD3 chains, such as CD3 zeta and CD3 epsilon chains.

In another embodiment, the method of expanding the T cells can furthercomprise isolating the expanded T cells for further applications. In yetanother embodiment, the method of expanding can further comprise asubsequent electroporation of the expanded T cells followed byculturing. The subsequent electroporation may include introducing anucleic acid encoding an agent, such as a transducing the expanded Tcells, transfecting the expanded T cells, or electroporating theexpanded T cells with a nucleic acid encoding a TCR, into the expandedpopulation of T cells, wherein the agent further stimulates the T cell.The agent may stimulate the T cells, such as by stimulating furtherexpansion, effector function, or another T cell function. In oneembodiment, the agent nucleic acid is co-electroporated with thechimeric membrane protein RNA. In another embodiment, the agent nucleicacid, such as a TCR RNA, is electroporated after culturing theelectroporated population. In a further embodiment, the agent nucleicacid, such as a TCR RNA, is electroporated into expanded T cells thatwere cryopreserved.

Therapy

The modified T cells described herein may be included in a compositionfor therapy. The composition may include a pharmaceutical compositionand further include a pharmaceutically acceptable carrier. Atherapeutically effective amount of the pharmaceutical compositioncomprising the modified T cells may be administered.

In one aspect, the invention includes a method for stimulating a Tcell-mediated immune response to a target cell or tissue in a subjectcomprising administering to a subject an effective amount of a modifiedT cell. In this embodiment, the T cell is modified as describedelsewhere herein. The modified T cells may be administered to inducelysis of the target cell or tissue, such as where the induced lysis isantibody-dependent cell-mediated cytotoxicity (ADCC).

In another aspect, the invention includes a method for adoptive celltransfer therapy comprising administering an effective amount ofpharmaceutical composition comprising the modified T cell describedherein to a subject in need thereof to prevent or treat an immunereaction that is adverse to the subject.

In yet another embodiment, a method of treating a disease or conditionassociated with enhanced immunity in a subject comprising administeringan effective amount of a pharmaceutical composition comprising themodified T cell described herein to a subject in need thereof.

The modified T cells generated as described herein can be uniform andpossess T cell function. Further, the modified T cells can beadministered to an animal, preferably a mammal, even more preferably ahuman, to suppress an immune reaction, such as those common toautoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis,multiple sclerosis, GVHD, enhancing allograft tolerance induction,transplant rejection, and the like. In addition, the cells of thepresent invention can be used for the treatment of any condition inwhich a diminished or otherwise inhibited immune response, especially acell-mediated immune response, is desirable to treat or alleviate thedisease. In one aspect, the invention includes treating a condition,such as an autoimmune disease, in a subject, comprising administering tothe subject a therapeutically effective amount of a pharmaceuticalcomposition comprising the modified T cell described herein.

Examples of autoimmune disease include but are not limited to, AcquiredImmunodeficiency Syndrome (AIDS, which is a viral disease with anautoimmune component), alopecia areata, ankylosing spondylitis,antiphospholipid syndrome, autoimmune Addison's disease, autoimmunehemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease(AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmunethrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiacsprue-dermatitis hepetiformis; chronic fatigue immune dysfunctionsyndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy(CIPD), cicatricial pemphigold, cold agglutinin disease, crest syndrome,Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoidlupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis,Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis,idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura(ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenilechronic arthritis (Still's disease), juvenile rheumatoid arthritis,Meniere's disease, mixed connective tissue disease, multiple sclerosis,myasthenia gravis, pernacious anemia, polyarteritis nodosa,polychondritis, polyglandular syndromes, polymyalgia rheumatica,polymyositis and dermatomyositis, primary agammaglobulinemia, primarybiliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena,Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis,scleroderma (progressive systemic sclerosis (PSS), also known assystemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome,systemic lupus erythematosus, Takayasu arteritis, temporalarteritis/giant cell arteritis, ulcerative colitis, uveitis, vitiligoand Wegener's granulomatosis.

The T cells generated as described herein can also be modified and usedto treat inflammatory disorders. Examples of inflammatory disordersinclude but are not limited to, chronic and acute inflammatorydisorders. Examples of inflammatory disorders include Alzheimer'sdisease, asthma, atopic allergy, allergy, atherosclerosis, bronchialasthma, eczema, glomerulonephritis, graft vs. host disease, hemolyticanemias, osteoarthritis, sepsis, stroke, transplantation of tissue andorgans, vasculitis, diabetic retinopathy and ventilator induced lunginjury.

In another embodiment, the modified T cell described herein may be usedfor the manufacture of a medicament for the treatment of an immuneresponse in a subject in need thereof.

Cells of the invention can be administered in dosages and routes and attimes to be determined in appropriate pre-clinical and clinicalexperimentation and trials. Cell compositions may be administeredmultiple times at dosages within these ranges. Administration of thecells of the invention may be combined with other methods useful totreat the desired disease or condition as determined by those of skillin the art.

The cells of the invention to be administered may be autologous,allogeneic or xenogeneic with respect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out inany convenient manner known to those of skill in the art. The cells ofthe present invention may be administered to a subject by aerosolinhalation, injection, ingestion, transfusion, implantation ortransplantation. The compositions described herein may be administeredto a patient transarterially, subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, byintravenous (i.v.) injection, or intraperitoneally. In other instances,the cells of the invention are injected directly into a site ofinflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.

The cells described herein can also be administered using any number ofmatrices. The present invention utilizes such matrices within the novelcontext of acting as an artificial lymphoid organ to support, maintain,or modulate the immune system, typically through modulation of T cells.Accordingly, the present invention can utilize those matrix compositionsand formulations which have demonstrated utility in tissue engineering.Accordingly, the type of matrix that may be used in the compositions,devices and methods of the invention is virtually limitless and mayinclude both biological and synthetic matrices. In one particularexample, the compositions and devices set forth by U.S. Pat. Nos.5,980,889; 5,913,998; 5,902,745; 5,843,069; 5,787,900; or 5,626,561 areutilized, as such these patents are incorporated herein by reference intheir entirety. Matrices comprise features commonly associated withbeing biocompatible when administered to a mammalian host. Matrices maybe formed from natural and/or synthetic materials. The matrices may benon-biodegradable in instances where it is desirable to leave permanentstructures or removable structures in the body of an animal, such as animplant; or biodegradable. The matrices may take the form of sponges,implants, tubes, telfa pads, fibers, hollow fibers, lyophilizedcomponents, gels, powders, porous compositions, or nanoparticles. Inaddition, matrices can be designed to allow for sustained release ofseeded cells or produced cytokine or other active agent. In certainembodiments, the matrix of the present invention is flexible andelastic, and may be described as a semisolid scaffold that is permeableto substances such as inorganic salts, aqueous fluids and dissolvedgaseous agents including oxygen.

A matrix is used herein as an example of a biocompatible substance.However, the current invention is not limited to matrices and thus,wherever the term matrix or matrices appears these terms should be readto include devices and other substances which allow for cellularretention or cellular traversal, are biocompatible, and are capable ofallowing traversal of macromolecules either directly through thesubstance such that the substance itself is a semi-permeable membrane orused in conjunction with a particular semi-permeable substance.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention may comprise themodified T cell as described herein, in combination with one or morepharmaceutically or physiologically acceptable carriers, diluents orexcipients. Such compositions may comprise buffers such as neutralbuffered saline, phosphate buffered saline and the like; carbohydratessuch as glucose, mannose, sucrose or dextrans, mannitol; proteins;polypeptides or amino acids such as glycine; antioxidants; chelatingagents such as EDTA or glutathione; adjuvants (e.g., aluminumhydroxide); and preservatives. Compositions of the present invention arepreferably formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented). Thequantity and frequency of administration will be determined by suchfactors as the condition of the patient, and the type and severity ofthe patient's disease, although appropriate dosages may be determined byclinical trials.

When “an immunologically effective amount”, “an anti-immune responseeffective amount”, “an immune response-inhibiting effective amount”, or“therapeutic amount” is indicated, the precise amount of thecompositions of the present invention to be administered can bedetermined by a physician with consideration of individual differencesin age, weight, immune response, and condition of the patient (subject).It can generally be stated that a pharmaceutical composition comprisingthe modified T cells described herein may be administered at a dosage of10⁴ to 10⁹ cells/kg body weight, preferably 10¹ to 10⁶ cells/kg bodyweight, including all integer values within those ranges. T cellcompositions may also be administered multiple times at these dosages.The cells can be administered by using infusion techniques that arecommonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng.J. of Med. 319:1676, 1988). The optimal dosage and treatment regime fora particular patient can readily be determined by one skilled in the artof medicine by monitoring the patient for signs of disease and adjustingthe treatment accordingly.

In certain embodiments, it may be desired to administer activated Tcells to a subject and then subsequently redraw blood (or have anapheresis performed), activate T cells therefrom according to thepresent invention, and reinfuse the patient with these activated andexpanded T cells. This process can be carried out multiple times everyfew weeks. In certain embodiments, T cells can be activated from blooddraws of from 10 ml to 400 ml. In certain embodiments, T cells areactivated from blood draws of 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml,80 ml, 90 ml, or 100 ml. Not to be bound by theory, using this multipleblood draw/multiple reinfusion protocol, may select out certainpopulations of T cells.

In certain embodiments of the present invention, cells expanded andmodified using the methods described herein, or other methods known inthe art where T cells are expanded to therapeutic levels, areadministered to a patient in conjunction with (e.g., before,simultaneously or following) any number of relevant treatmentmodalities, including but not limited to treatment with agents such asantiviral therapy, cidofovir and interleukin-2, Cytarabine (also knownas ARA-C) or natalizumab treatment for MS patients or efalizumabtreatment for psoriasis patients or other treatments for PML patients.In further embodiments, the T cells of the invention may be used incombination with chemotherapy, radiation, immunosuppressive agents, suchas cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506,antibodies, or other immunoablative agents such as CAM PATH, anti-CD3antibodies or other antibody therapies, cytoxin, fludaribine,cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. These drugs inhibit either the calciumdependent phosphatase calcineurin (cyclosporine and FK506) or inhibitthe p70S6 kinase that is important for growth factor induced signaling(rapamycin). (Liu et al., Cell 66:807-815, 1991; Henderson et al.,Immun. 73:316-321, 1991; Bierer et al., Curr. Opin. Immun. 5:763-773,1993). In a further embodiment, the cell compositions of the presentinvention are administered to a patient in conjunction with (e.g.,before, simultaneously or following) bone marrow transplantation, T cellablative therapy using either chemotherapy agents such as, fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodiessuch as OKT3 or CAMPATH. In another embodiment, the cell compositions ofthe present invention are administered following B-cell ablative therapysuch as agents that react with CD20, e.g., Rituxan. For example, in oneembodiment, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. Incertain embodiments, following the transplant, subjects receive aninfusion of the expanded immune cells of the present invention. In anadditional embodiment, expanded cells are administered before orfollowing surgery.

The dosage of the above treatments to be administered to a patient willvary with the precise nature of the condition being treated and therecipient of the treatment. The scaling of dosages for humanadministration can be performed according to art-accepted practices. Thedose for CAMPATH, for example, will generally be in the range 1 to about100 mg for an adult patient, usually administered daily for a periodbetween 1 and 30 days. The preferred daily dose is 1 to 10 mg per dayalthough in some instances larger doses of up to 40 mg per day may beused (described in U.S. Pat. No. 6,120,766).

It should be understood that the method and compositions that would beuseful in the present invention are not limited to the particularformulations set forth in the examples. The following examples are putforth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use the cells,expansion and culture methods, and therapeutic methods of the invention,and are not intended to limit the scope of what the inventors regard astheir invention.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook,2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of AnimalCells” (Freshney, 2010); “Methods in Enzymology” “Handbook ofExperimental Immunology” (Weir, 1997); “Gene Transfer Vectors forMammalian Cells” (Miller and Calos, 1987); “Short Protocols in MolecularBiology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles,Applications and Troubleshooting”, (Babar, 2011); “Current Protocols inImmunology” (Coligan, 2002). These techniques are applicable to theproduction of the polynucleotides and polypeptides of the invention,and, as such, may be considered in making and practicing the invention.Particularly useful techniques for particular embodiments will bediscussed in the sections that follow.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out the preferred embodiments ofthe present invention, and are not to be construed as limiting in anyway the remainder of the disclosure.

The materials and methods employed in these experiments are nowdescribed.

Primary human lymphocytes. Primary lymphocytes were stimulated withmicrobeads coated with CD3 and CD28 stimulatory antibodies (LifeTechnologies, Grand Island, N.Y., Catalog) as described (Human genetherapy 2011, 22(12):1575-1586). T cells were cryopreserved at day 10 ina solution of 90% fetal calf serum and 10% dimethylsulfoxide (DMSO) at1×10⁸ cells/vial.

NALM-6 was purchased from the German DSMZ Cell Collection (DSMZ catalogcode: ACC 128). K562 and PC3 were purchased from American Type CultureCollection. 624mel melanoma line was obtained from the Surgery Branch(NCI/NIH). All the cell lines were cultured as instructed and routinelytested for mycoplasma contamination and confirmed as being negative.

Generation of TCR constructs for mRNA electroporation and lentiviraltransduction. 1G4 NY-ESO-1 TCR with different mutations (1G4 and 8F) andCARs (PSCA or CD19) were synthesized and/or amplified by PCR, based onsequencing information provided by the relevant publications (TheJournal of experimental medicine 2005, 201(8):1243-1255; J Immunol 2008,180(9):6116-6131), and subcloned into pGEM.64A RNA based vector or pTRPElentiviral vectors.

Human primary T cell preparation. Primary human CD4 and CD8 T cells wereisolated from healthy volunteer donors following leukapheresis bynegative selection using RosetteSep kits (Stem Cell Technologies,Vancouver BC, Canada). All specimens were collected under a UniversityInstitutional Review Board-approved protocol, and written informedconsent was obtained from each donor.

Design and construction of CRISPRs. Cas9 DNA was synthesized by PCR theninserted to PGEM vector. gRNAs were selected by GN19 with a NGG PAMsite, with some selected from N20 with a NGG PAM site. All gRNAscontained complementary sequences comprised of more than 13 base pairmispairs, with potential off-target mRNA sites excluded (Table 1). GRNAswere designed, as shown in FIG. 1A, and synthesized by overlap PCR. AllgRNA PCR products were ligated into the MSGV vector. In vitrotranscribed CAS9 and gRNA targeted constant regions of TCR α, β chainsand beta-2 microglobin. gRNAs were designed to target either a sequencewithin exon 1 of the TCR α constant region, a consensus sequence commonto exon 1 of both TCR β constant regions 1 and 2, beta-2 microglobulinor PD1. Sequences encoding the gRNAs were assembled using overlap PCRand cloned into the MSGV vector containing a T7 promoter. These plasmidswere linearized with EcoRI. gRNA was in vitro transcribed. Cas9 mRNA wasin vitro transcribed using mMESSAGE mMACHINE T7 ULTRA kit (LifeTechnologies, Carlsbad, Calif.). The mRNA was stored at −80° C. innuclease-free vials for single use. The gRNA targeting sequences usedfor the animal study were as follows:

TRAC-gRNA: SEQ ID NO: 1 TGTGCTAGACATGAGGTCTA, TRBC-gRNA: SEQ ID NO: 2GCAGTATCTGGAGTCATTGA, B2M-gRNA: SEQ ID NO: 3 CGCGAGCACAGCTAAGGCCA,PD1-gRNA: SEQ ID NO: 4 GGCGCCCTGGCCAGTCGTCT, FAS-gRNA: SEQ ID NO: 5GAGGGTCCAGATGCCCAGCA,

Flow cytometry. The following monoclonal antibodies and reagents wereused with indicated specificity and the appropriate isotype controls.From BD Biosciences (San Jose, Calif.): APC-conjugated anti-CD3(555335), FITC-anti-CD8(555366), PE-anti-CD8(555635), FITC-anti-CD27(555440), PE-anti-CD107(555801), PE-anti-beta-2 microglobin (551337),FITC-anti-HLA (555552); Biolegend (San Diego, Calif.). FITC-anti-CD45RO(304204), APC-anti-CD62L (304814), APC-anti-CCR7(353214); and BeckmanCoulter (Pasadena, Calif.): PE-anti-Vb13.1 (IM2021U). Data was acquiredon a FACS Accuri (BD Biosciences, San Jose, Calif.) using CellQuestversion 3.3 (BD Biosciences, San Jose, Calif.) and analyzed by FCSExpress version 3.00 (De Novo Software, Los Angeles, Calif.) or FlowJoversion 7.6.1 (Tree Star, Inc. Ashland, Oreg.).

Propagation of primary T cells. Primary human T cells were cultured inRPMI 1640 supplemented with 10% FCS, 100-U/ml penicillin, 100-g/mlstreptomycin sulfate, 10-mM Hepes, and stimulated with magnetic beadscoated with anti-CD3/anti-CD28 at a 1:3 cell to bead ratio. Cells werecounted and fed every 2 days and once T cells appeared to rest down, asdetermined by both decreased growth kinetics and cell size, the T cellswere either used for functional assays or cryopreserved.

Generation of CD3^(neg) T cells. DNA supercoiled plasmids werelinearized by SpeI and EcoRI, respectively. gRNA was in vitrotranscribed by T7 mScript™ Standard mRNA Production System (Cambio,C-MSC100625, Cambridge, England). All mRNA (Cas9, TCR α, TCR β and CARs)was in vitro transcribed using mMESSAGE mMACHINE T7 ULTRA kit (LifeTechnologies, AM1345, Carlsbad, Calif.). T cells were stimulated byCD3/CD28 dynabeads for three days prior to electroporation. Ten millionprimary T cells were de-beaded prior to electro-transfer of 20 μg Cas9,10 μg gRNA species into the cells with a 360V, 1 ms parameter by BTX830,following a second and/or a third electro-transfer of 10 μg gRNA. Also,T cells were washed three times with OPTI-MEM and re-suspended inOPTI-MEM (Invitrogen) at a final concentration of 1-3×10⁸ cells/ml.Subsequently, 0.1 ml of the cells was mixed with 10 μg of IVT RNA (or asindicated) and electroporated in a 2 mm cuvette. Ten million primary Tcells were de-beaded prior to the electrotransfer of 20 μg of Cas9 and10 μg of gRNA species into the cells using a BTX830 (Harvard ApparatusBTX) at 360 V and 1 ms; this process was followed by a second and athird electrotransfer of 5 μg of gRNA 12 to 24 hours later.

Following electroporation, cells were immediately placed in 2 mL ofpre-warmed culture media and cultured at 37° C., 5% CO₂, or 32° C., 5%CO₂ for 1 day then returned to 37° C., 5% CO₂.

TCR α and β double disruption or TRAC, TRBC and B2M triple disruption.To generate TCR α and β double-knockout T cells, Cas9 mRNA wasco-electroporated with two different gRNAs targeting TCR α chain (TRAC),and TCR β chain (TRBC). The TCR α and β double-knockout T cells could bepurified in 2 steps: 1) depletion of TCR-positive and a chainsingle-knockout cells with anti-CD3 microbeads after the electroporationof the 1G4 TCR α chain RNA, and 2) depletion of TCR β chainsingle-knockout cells with anti-CD3 microbeads after the electroporationof the TCR β chain RNA. For TRAC, TRBC and B2M triple disruption, Tcells were electroporated with Cas9 mRNA and gRNAs targeting the TCR αand β chains and beta-2 microglobulin 3 days after anti-CD3/CD28 beadstimulation. The HLA-I-negative cell population was enriched on day 9and electroporated with TCR α chain RNA. The TCR-negative population wasenriched on day 10. Five days later, these cells were electroporatedwith TCR β chain RNA, and the TCR-negative cell population was sortedthe next day to obtain universal T cells. On day 18, TCR or CAR RNA waselectroporated into the universal T cells to generate universal effectorcells. TCR and HLA-I molecule expression was confirmed at each step.

Generation of universal CART cells. Universal CART cells were generatedby combing the lentiviral transduction of CD19 or PSCA CAR with the RNAelectroporation of CRISPR/gRNAs. 1 day after anti-CD3/CD28 beadsstimulation, T cells were transduced with lentiviral-CD19 or PSCA CAR. 2days later, Cas9 and gRNAs targeting TCR α, β chain, B2M, PD1 weretransferred into T cells by electroporation. 6 days after CRISPRsdelivery, T cells negative for CD3, HLA-I, PD1 were sorted by microbeadsdepletion.

Enrichment of CD3^(neg) T cells. Cells washed with Auto MACS buffer wereincubated for 30 minutes with CD3 microbeads (Miltenyi Biotec,130-050-101, Auburn, Calif.) at 4° C. After washing twice, cells werepassed through a LD column (MiltenyiBiotec, 130-042-901, Auburn,Calif.), and the flow-through fraction was collected for further use.The CD3 expression of CD3^(neg) T cells was restored byco-electroporation of 1G4TCR α and □ mRNA, and the cells were expandedusing a single Rapid Expansion Protocol (REP), CD3/CD28 Dynabeads orK562-based aAPC.

Generation and propagation of CD3^(neg) T cells. CD3^(neg) T cells hadCD3 expression restored by electro-transfer of exogenous 1G4TCR alphachain and TCR beta chain in vitro transcribed mRNA (5 μg for eachchain). These cells were expanded using a single Rapid ExpansionProtocol (REP). PBMCs from three different donors: ND052 105×10⁶, ND40583×10⁶, ND410 136×10⁶, were irradiated, then mixed together, to obtain atotal of 324×10⁶ PBMCs. The PBMCs were re-suspended in a final volume of90 ml then R10 were added to 300 ml, mixed, and divided into two T150 mlflasks. OKT were added to a final concentration of 30 ng/ml. On day 2,IL-2 was added to 50 CU/ml. From day 5, cells were counted and fed every2 days and once T cells appeared to rest down, as determined by bothdecreased growth kinetics and cell size, they were either used forfunctional assays or cryopreserved.

Sanger sequencing. The level of genomic disruption of TCR α chain(TRAC), TCR β chain 1 (TRBC1), and TCR β chain 2 (TRBC2) in T cells wasdetermined by Surveyor Nuclease assay (Transgenomics, Omaha, Nebr.). Thepercent target disruption was quantified by densitometry. The PCRprimers used for the amplification of target locus were:

TRAC forward, SEQ ID NO: 6 5′-TCATGTCCTAACCCTGATCCTCTT-3′ TRAC reverse,SEQ ID NO: 7 5′-TTGGACTTTTCCCAGCTGACAGA-3′ TRBC total forward,SEQ ID NO: 8 5′-TACCAGGACCAGACAGCTCTTAGA-3′ TRBC total reverse,SEQ ID NO: 9 5′-TCTCACCTAATCTCCTCCAGGCAT-3′

PCR products were purified and ligated to TOPO cloning vector(Invitrogen) then transformed in E. coli. Single clone was picked andsequenced to calculate the indels.

Generation of siRNA and CRISPRi for electroporation. RNA duplextargeting TCR constant regions for either alpha(5′-rArGrGrArGrGrArUrUrCrGrGrArArCrCrCrArArUrCrArCrUrGrArC-3′ SEQ IDNO:10 and 5′-rCrArGrUrGrArUrUrGrGrGrUrUrCrCrGrArArUrCrCrUrCCT-3′ SEQ IDNO:11) or beta(5′-rArCrCrUrCrCrUrUrCrCrCrArUrUrCrArCrCrCrArCrCrArGrCrUrC-3′ SEQ IDNO:12 and 5′-rGrCrUrGrGrUrGrGrGrUrGrArArUrGrGrGrArArGrGrArGGT-3′ SEQ IDNO:13) were designed using Custom RNAi Design Tool (Integrated DNATechnologies, Coralville, Iowa) and the siRNA was synthesized(Integrated DNA Technologies, Coralville, Iowa). siRNA for both TCRalpha and beta was mixed and electroporated into stimulated T cells forendogenous TCR knockdown.

mRNA in vitro transcription and T cell electroporation. T7 mscriptsystems kit (CellScript) was used to generate in vitro transcribed (IVT)RNA. CD3/CD28 bead stimulated T cells were electroporated with IVT RNAusing BTX EM830 (Harvard Apparatus BTX) as previously described (Cancerresearch 2010, 70(22):9053-9061). Briefly, T cells were washed threetimes and resuspended in OPTI-MEM (Invitrogen) at a final concentrationof 1-3×10⁸ cells/ml. Subsequently, 0.1 ml of cells were mixed with 10 ugIVT RNA (or as indicated) and electroporated in a 2 mm cuvette.

ELISA assays. Target cells, different tumor cell lines expressing CD19,were washed and suspended at 1×10⁶ cells/ml in R10 medium (RPMI 1640supplemented with 10% fetal calf serum; Invitrogen). 100 ul of eachtarget cell type was added in duplicate to a 96 well round bottom plate(Corning). Effector T cells were washed, and re-suspended at 1×10⁶cells/ml in R10 medium and then 100 ul of T cells were combined with thetarget cells in the indicated wells. In addition, wells containing Tcells alone were prepared as a control. The plates were incubated at 37°C. for 18 to 20 hours. After the incubation, supernatant was harvestedand subjected to an ELISA assay (eBioscience).

CD107a staining Cells were plated at an Effector cell:T cell ratio of1:1 (1×10⁵ effectors to 1×10⁵ targets) in 160 μl of complete RPMI mediumin a 96 well plate. 20 μl of phycoerythrin-labeled anti-CD107a antibody(BD Biosciences, 555801) was added and the plate was incubated at 37° C.for 1 hour before adding Golgi Stop (2 ul Golgi Stop in 3 ml RPMImedium, 20 ul/well; BD Biosciences, 51-2092KZ) and incubating the platefor another 2.5 hours. Then 5 μl FITC-anti-CD8 and 5 ul APC-anti-CD3were added and incubated at 37° C. for 30 min. After incubation, thesamples were washed with FACS buffer and analyzed by flow cytometry.

Luciferase based CTL assay. Naml6-CBG tumor cells were generated andemployed in a modified version of a luciferase based cytotoxic Tlymphocyte assay. Briefly, click beetle green luciferase (CBG) wascloned into the pELNS vector, packaged into lentivirus, transduced intoNaml6 tumor cells and sorted for CBG expression. The resulting Naml6-CBGcells were washed and resuspended at 1×10⁵ cells/ml in R10 medium, and100 ul of CBG-labeled cells were incubated with different ratios of Tcells (e.g. 30:1, 15:1, etc) overnight at 37° C. 100 ul of the mixturewas transferred to a 96 well white luminometerplate. 100 ul of substratewas added to the cells and luminescence was immediately determined. Theresults are reported as percent killing based on the luciferase activityin the wells with tumor cells but no T cells (% killing=100−((RLU fromwell with effector and target cell coculture)/(RLU from well with targetcells)×100)).

Mouse xenograft studies. Studies were performed as previously describedwith certain modifications (Human gene therapy 2011, 22(12):1575-1586;Proceedings of the National Academy of Sciences of the United States ofAmerica 2009, 106(9):3360-3365). Briefly, 6-10 week old NOD/SCID gamma(NSG) mice were injected subcutaneously with 1×10⁶ PC3-CBG tumors cellson the right flank at day 0 and the same mice were given SK-OV3-CBGtumor cells (5×10⁶ cells/mouse, subcutaneously.) on the left flank atday 5. The mice were treated with T cells via the tail vein at day 23post PC3-CBG tumor inoculation, such that both tumors were approximately200 mm³ in volume. Lentivirally transduced T cells were given at 1×10⁷cells/mouse (10M), or 3×10⁶ cells/mouse (3M). Briefly, for the Nalm6tumor model, 6- to 10-week-old NOD/SCID gamma (NSG) mice were injectedwith 1×10⁶ click beetle green (CBG) transduced Nalm6 (Nalm6-CBG) cellsthrough the tail vein on day 0. The T cell treatment began on day 7after the tumor inoculation. For the PC3-PDL1 solid tumor model, 6- to10-week-old NOD/SCID gamma (NSG) mice were injected subcutaneously with1×10⁶ PSCA, PD-L1 and CBG transduced PC3 (PC3-PSCA-PDL1-CBG) tumorscells in the right flank on day 0. The mice were treated with T cellsvia the tail vein at day 22 post PC3-PDL1-CBG tumor inoculation, suchthat the tumors were approximately 200 mm³ in volume. T cells were givenat 2×10⁶ cells/mouse (2M). Animals were randomized and grouped based onbaseline tumor size. All animals were included in the experiments andblinded tumor assessment was done for all the animal experimentsconducted.

T cell stimulation, lentiviral transduction and CRISPR electroporationprocedure. FIG. 84 shows the procedure used to stimulate, lentiviraltransduce and CRISPR electroporate T cells. On day 0, T cells wereobtained from 3 donors (100×10⁶ cells/donor). The cells were stimulatedwith anti-CD3/anti-CD28 beads at a T cell:bead ratio of 1:3. Theconcentration of cells was adjusted to 0.5×10⁶/ml with 100 mL/flask. Onday 1, stimulated T cells were transduced with CD19 CAR lentivirus atmultiplicity of infection (MOI) of 2. 50 mL (25×10⁶ cells) of T cellswere reserved as unmodified T cells (Group 9). On Day 3, the beads wereremoved, the cells washed 2× in Opti-MEM media, and the transduced Tcells from each donor were separated into two groups, CART/mock EP (10mL, 50×10⁶/mL) and CART/CRISPR (10 mL, 50×10⁶/mL). The cells were thenelectroporated with CAS9 RNA (1st EP) at 500V/1 ms with 120 μg of CAS9RNA/400 μL of T cells. After electroporation, Groups 1, 3, 5 and 7 cellswere then split by culturing T cells in half new medium and halfcultured medium. On day 4, the cells were washed twice and resuspendedin Opti-MEM at 50×10⁶/mL. 20 μg TRBC4 and B2M gRNA was electroporatedinto the 400 μL of T cells. After electroporation, the cells werecultured at 1×10⁶ cells/mL in half fresh medium and half culturedmedium. On days 5 and 7, the cells were split and resuspended in halffresh medium and half cultured medium. On day 8, CD3+ cells were removedfrom Groups 2, 4 and 6 via a low-density column. The CD3-T cells wereresuspended at 0.5-1×10⁶ cells/mL in half fresh medium and half culturedmedium and cultured to expand the cells. On day 11, the T cells wereharvested and 25×10⁵ cells from the three donors were sent forkaryotyping. The remaining cells were aliquoted and frozen.

The results of the experiments are now described.

Example 1: Disruption of the TCR-CD3 Complex on T Cells Using CRISPR

Thirteen gRNAs targeting the constant regions of TCR α chain, 10 gRNAstargeting the constant regions TCR β chain, and 10 RNAs targeting thebeta-2 microglobin gene. (FIGS. 1A-1C and FIGS. 9A-9D) were developedand tested in 293T cells. Primary human T cells were propagated ex vivofor three days with anti-CD3/anti-CD28 dynabeads for three days. Sincetransient expression of CRISPR is sufficient to mediate gene knockout, a“hit-and-run” delivery strategy was developed to transiently express theCRISPRs by utilizing electro-transfer of in vitro transcribed RNAencoding CAS9 and gRNAs (FIG. 2C).

To measure TCR expression, a mAb specific for CD3 was used, which isonly present on the cell surface when TCR antibody is expressed. Sixdays after electro-transfer, flow cytometric analysis revealed thatCRISPRs targeting TRBC eliminated CD3 expression on primary T cells atlevels of 13.7 (FIG. 2D) in donor ND147. The efficiency of TCR knockoutcorrelated with the amount of electro-transferred mRNA (FIG. 2D).Although the electro-transfer of RNA in primary T cells waswell-tolerated, a slight reduction in cell viability was observed thatcorrelated with increasing amounts of introduced RNA. ZFN and TALENmediated gene disruption has been reported to be more efficient whencells were transiently exposed to mild hypothermia. The same phenomenonwas observed with this CRISPR system.

The T cells were cultured for 1 day at 32° C. after electro-transfer.CRISPR-mediated disruption of CD3 was up to 2.5-fold better whenelectroporated T cells were cultured at 32° C. versus 37° C. Using thisapproach, 5.43% and 16.7% of electroporated T-cells lost expression ofCD3 using the CRISPRs targeting TRAC and TRBC, respectively, (FIG. 2D,lower panel). No change in the levels of CD3 negative cells in the CAS9MOCK samples and no appreciable decrease in viability (measured byTrypan blue) were observed.

When gRNAs were electro-transferred for a second and a third time, theefficiency of eliminating CD3 expression on primary T cells at levelswas greatly improved.

-   -   Targeting TRAC: at levels reaching 77% after three times        electro-transfer of gRNA (FIG. 4A),    -   Targeting TRAC or TRBC at levels reaching 64.5% or 57.5%,        respectively, after a second electro-transfer of gRNAs with a        slight decreased viability (FIG. 4C).

To confirm that electroporated T cells had been genetically modified atthe intended gRNA target sites (TCR α or β loci), Sanger sequencing wasperformed using specific oligonucleotide primers flanking target siteswithin TRAC, TRBC1, or TRBC2. Multiple peaks at the indicated PCRproducts starting from the target sites were present only afterelectro-transfer of CRISPRs and the percent disruption correlated withloss of cell surface CD3 expression (FIGS. 1C and 3B). These experimentsin primary T cells confirmed that CRISPRs designed to target TRAC orTRBC led to permanent disruption of αβ TCR expression, as assessed bySanger sequencing and confirmed by flow cytometric analysis of CD3.

Example 2: Enrichment of TCR αβ Negative T Cells

For future clinical applications, rapid and robust methods for isolatingsources of TCR disrupted populations may be utilized. To begin toaddress this issue, the TCR/CD3^(neg) population was enriched bynegative selection using clinically-approved paramagnetic beads and adepletion column. With a single depletion step, the CD3^(neg),population was enhanced to over 99% (FIG. 3A). A CD3^(neg) populationcould not be enriched from untransfected control cells. Back-to-backdepletion steps resulted in >99% enrichment, without skewing the CD4 orCD8 T cell subsets (FIG. 3C). Sequencing results also showed deletionsand insertions were introduced to TCR alpha and beta locus after CRISPRmodification (FIG. 3D).

Example 3: Generation of HLA-CLASS I^(neg) T Cells by CRISPR

To test the ability of CRISPR to knock out HLA-CLASS I expression fromallogeneic T cells, gRNAs targeting beta-2 microglobin were designed.The beta-2 microglobin locus could be manipulated by CRISPR in 293 Tcells (FIG. 9A). Evidence showed disruption of beta-2 microglobinabolished T cell surface HLA-CLASS I expression (FIG. 9B).

IFN-gamma greatly improved, approximately 10 fold, targeting efficiencyof beta-2 microglobin in T cells (FIG. 9C). Multiple electro-transfersof beta-2 microglobin gRNAs gave a 66% beta-2 microglobin negativepopulation (FIG. 11A).

For future allograft transplantation clinical applications, rapid androbust methods for isolating sources of HLA-CLASS I null populationswill be needed. To begin to address this issue, the cells were labeledwith PE-anti-beta-2 microglobin antibody, and enriched for a HLA-CLASSI^(neg), population by negative selection using clinically-approvedparamagnetic anti-PE microbeads and a depletion column. With a singledepletion step, the HLA-CLASS I^(neg) population was enhanced to over99%. A HLA-CLASS I^(neg) population could not be enriched fromuntransfected control cells. An analysis of HLA-CLASS I repertoire inenriched HLA-CLASS I^(neg) T cells via flow cytometry validated theelimination of HLA-CLASS I expression from the cell surface (FIG. 9D).

Example 4: CD3^(neg) T Cells can be Propagated by Different Methods

CD3^(neg) T cells restored CD3 expression after electro-transfer ofexogenous 1G4-TCR alpha and beta chain in vitro transcribed mRNA (5 μgeach). These cells were expanded by: (1) a single Rapid ExpansionProtocol (REP), then tested for activity and specificity. PBMCs wereobtained from three different donors: ND052 105×10⁶, ND405 83×10⁶, ND410136×10⁶. Cell were after irradiated, then mixed together, and a total324×10⁶ PBMCs were obtained. 2×10⁶ cells were electro-transferred withRNA. CD3^(neg) T were re-suspended in a final volume of 90 ml and R10media was added for a total volume of 300 ml. The cells were dividedinto 2 T150 ml flasks. OKT was added to a final concentration of 30ng/ml. On day 2, IL-2 was added to 50 CU/ml. From day 5, cells werecounted and fed every 2 days and once T cells appeared to rest down, asdetermined by both decreased growth kinetics and cell size, they wereeither used for functional assays or cryopreserved.

After a single REP, CD3^(neg) T cells were expanded for a 500 foldincrease in number. These cells were expanded by: (2) stimulated withmagnetic beads coated with anti-CD3/anti-CD28 at a 1:3 cell to beadratio.

After a single REP, CD3^(neg) T cells were expanded for a 500 foldincrease in number. These cells were expanded by: (3) co-cultured withirradiated K562-CD19 and K562/86/64/A2(2D11) in equal mixture at aconcentration of 1×10⁶/ml.

After a single REP, CD3^(neg) T cells were expanded for a 500 foldincrease in number. These cells were expanded by: (4) co-cultured withirradiated K562-CD19 and K562/86/64/A2(2D11) in equal mixture at aconcentration of 1×10⁶/ml with 30 ng/ml OKT

After a single REP, CD3^(neg) T cells were expanded for a 500 foldincrease in number. These cells were expanded by: (5) co-cultured withirradiated K562-CD19 and K562/86/64/A2(2D11) in equal mixture at aconcentration of 1×10⁶/ml with 1 mg/ml NY-ESO peptide.

Example 5: Re-Direction of TCR^(neg) T Cells by Electro-Transfer of TCR

To test the function of TCR^(neg) T cells, these cells were re-directedby electro-transfer of TCR. By introducing TCR alpha chain and TCR betachain, these cells expressed high levels of TCR. The expression ofVb13.1 was much higher in electro-transferred TCR^(neg) T cells comparedto CAS9 MOCK control (FIG. 7A). When the cells were co-cultured with theNalm-6 NY-ESO leukemia cell line, positive for both HLA-A2 and NY-ESO,the cells showed high levels of 107a, indicating elevated de-granulationactivity (FIG. 7B). The killing assay also showed potent toxicitytowards this cell line (FIG. 7C). This indicated that these cells arepotentially safer than traditional clinical trials with T cellexpressing CARs and TCRs, as these cells would not trigger GVHD and haveless miss-pair toxicity than normal T cells with TCR treatment.

Some reports have shown that T cells can be genetically edited by ZFNsor TALEN to eliminate expression of the endogenous αβ TCR. The methodsand compositions described herein to selectively deplete T cellsexpressing undesired αβ TCR also include incomplete knockout of theendogenous TCR to treat GVHD and inhibit endogenous TCR from adverselyaffecting CAR function (e.g., through competition for transcriptionfactors). Therefore, a genetic approach was designed using designer ZFNsto permanently disrupt the α and β constant region sequences in T cells,thereby eliminating TCR expression.

ZFNs and TALENs are artificial restriction enzymes generated by fusing aDNA binding domain to a DNA cleavage domain. When ZFNs and TALENs do notwork efficiently, it is often difficult to determine the cause. Failurecould reflect a problem with the design, with accessibility of thetarget sequence, or a delivery issue. At the same time, ZFN targetingefficiency is usually low in T cells, making it difficult to manipulatemultiple genes at one time.

Distinct ZFNs and TALENs, the CRISPR/Cas system has recently emerged asa potentially facile and efficient alternative to ZFNs and TALENs forinducing targeted genetic alterations. Recent work has shown that targetrecognition by the Cas9 protein requires a ‘seed’ sequence within thecrRNA and a conserved di-nucleotide-containing protospacer adjacentmotif (PAM) sequence upstream of the crRNA-binding region. TheCRISPR/CAS system can thereby be retargeted to cleave virtually any DNAsequence by redesigning the crRNA. The data disclosed herein shows thepotential for gene editing by CRISPR/CAS in 293T cells and primary Tcells. The CRISPR/CAS system can simultaneously target multiple genomicloci by co-expressing a single CAS9 protein with two or more gRNAs,making this system uniquely suited for multiplex gene editing orsynergistic activation of target genes. By administering different gRNAstogether with CAS9, multiple genes can be simultaneously disrupted in Tcells.

Example 6: HLA CLASS I and TCR α, β Chain Triple Knockout by CRISPR

To work toward “off-the-shelf” allogeneic t-cell therapies formalignancies and infectious diseases, cell therapy by infusion of Tcells was designed to reconstitute immunity against pathogens andmalignancies. The amount of time required to manufacture T cells withthe desired properties and in sufficient numbers ex vivo is oftenincompatible with the treatment window for patients. Furthermore,autologous T cells from patients with advanced disease may havecompromised function and be tolerant to desired antigens.

To address this, patients can be infused with allogeneic T cells toavoid immune-mediated rejection caused by host T cells recognizingdisparate major or minor histocompatibility antigens on the infusedcells. To broaden the application of T cell therapy, and for futureallograft transplantation, rapid and robust methods for isolatingsources of TCR and HLA-CLASS I disrupted populations can be generated.

ZFN and TALEN comprise a zinc finger DNA-binding domain designed to binda specific DNA sequence fused to the cleavage domain of Foklendonuclease. The design and construction of ZFN and TALEN is verycomplicated and time consuming if there is more than one gene to bemanipulated, because the genes must be targeted individually. With theCRISPR system described herein, the efficiency and shortened the timecourse of gene disruption can be obtained.

To address this issue, CAS9 was electro-transferred with three differentgRNAs targeting TRAC, TRBC and beta-2 microglobin. Cells were labeledwith PE-anti-beta-2 microglobin antibody and enriched for a HLA-CLASSI^(neg) population by negative selection using clinically-approvedparamagnetic anti-PE microbeads and a depletion column. With a singledepletion step, the HLA-CLASS I^(neg) population was enhanced to over99% (FIG. 9D). Then the cells were re-introduced with TCR alpha chain,and HLA-CLASS I^(neg) CD3^(neg) population was enriched by microbeads(FIG. 11). Five days later, the TCR beta chain was re-introduced intothe cells, and a HLA-CLASS I^(neg) CD3^(neg) population was enriched bymicrobeads again. Two days later, TCR was electro-transferred into thesetriple knock out cells. On the day after electro-transformation, thecells were stimulated with CD3/CD28 dynabeads. Then, the cells underwentlentiviral delivery of antigen specific TCR the next day and cultureexpansion.

Example 7: FAS, PD1, CTLA4, PPP2R2D Knockout by CRISPR

The FAS receptor/FAS ligand (FAS/FASL) apoptosis signaling pathway hasbeen widely studied and is well characterized in T cells. PD1 and CTLA4are two major inhibitory signaling pathway in T cells that have alsobeen extensively studied. Direct evidence for the potential therapeuticimpact of targeting these pathways came from studies in preclinicalmurine tumor models demonstrating enhanced anti-tumor immunity afterantibody-mediated blockade of CTLA-4, PD-1 or PD-L1. Similar antibodiesfor use in humans have been developed, and early clinical data showedpromising results. Ppp2r2d knockdown may also inhibit T-cell apoptosisand enhance T-cell proliferation, as well as cytokine production.Ppp2r2d has potential as a target to improve the function of human Tcells.

To address this issue, CAS9 and three different gRNAs targeting FAS,PD1, CTLA4, PPP2r2d were electro-transferred into T cells. Sangersequencing data showed that the indicated locus of FAS, PD1, CTLA4,PPP2r2d had been modified by the CRISPRs. FAS was also replaced by GFPwith homologous recombination triggered by CRISPR. FACS data showed thesurface expression of FAS and PD1 was abolished.

Example 8: Generation of IPS Cells with Gene Modified Primary and TCells

Progress in adoptive T-cell therapy for cancer and infectious diseasesis hampered by the lack of readily available and antigen-specific humanT lymphocytes. Pluripotent stem cells could provide an unlimited sourceof T lymphocytes. To address this issue, the expression of FAS, PD1,CTLA4, PPP2r2d were disrupted in primary cells and T cells.

Sendai virus was used to reprogram primary cells and T cells. There aremultiple methods to generate iPSCs, including virus-mediated genetransduction and chemical induction. While lentiviral and retroviralvectors require integration into host chromosomes to expressreprogramming genes, DNA-based vectors, such as adenovirus,adeno-associated virus, and plasmid vectors, exist episomally and do notrequire integration. However, they may still be integrated into hostchromosomes at certain frequencies, and the reprogramming efficiency isrelatively low. Likewise, mRNA based reprogramming is complicated andshown to be extremely inefficient.

Unlike these methods, Sendai virus does not integrate into the hostgenome or alter the genetic information of the host cell. Sendai virusalso has reprogramming potential comparable to lentiviral- andretroviral-based gene transduction.

Each well in a 24 well plate was seeded with 0.1 million wild type,FAS^(neg), CD3^(neg) TCR alpha chain and TCR beta chain knock-out Tcells. The cells were stimulated with CD3/CD28 beads. At day 3 poststimulation, the beads were removed, the cells resuspended in 1 mL ofpre-warmed T cell complete medium, and then incubated with a calculatedvolume of CytoTune Sendai virus comprising a polycistronic vector forexpression of hKlf4, hOct3/4 and hSox2 in the cells (Lifetechnologies,Carlsbad, Calif.). Treated T cells were seeded in 24 well plates, andcentrifuged at 2250 rpm for 90 minutes at room temperature. Anadditional 1 mL of complete T cell medium was added to each well and theplate was incubated overnight at 37° C. in a humidified atmosphere of 5%CO2.

On the day after transduction, Sendai virus was removed by washing the Tcells with fresh complete medium and culturing the cells for 2 days.Media was half changed every day. On day 3 after infection, cells weretransferred to MEF feeder plates and cultured in T cell medium withoutany cytokines. Four days after infection, the cells were cultured instandard hES medium. Media was changed every day. ES-like colonies wereobserved around day 7. The cells were cultured in conditioned hES mediumfrom day 15 and cultures continued for an additional 10 days. Colonieswere picked at around 25 to 30 days after transduction.

At around day 4, cell clumps were formed on feeder cells, indicatingthat the initiation of the reprogramming process. T cells went throughdramatic morphological changes during the process of reprogramming toiPSCs. At around day 12, large cell clumps with loose edges began toemerge. At around day 18, T cells were transformed to typical ES-likecolonies with well defined edges. Typical embryonic stem cell morphologywas observed indicating that the FAS^(neg), CD3^(neg) TCR alpha chainand TCR beta chain knock-out T cells were induced to a pluripotent stateunder defined reprogramming conditions (FIGS. 17A and 18A).

FAS^(neg) T cells were easier to reprogram to iPSCs, at an efficiency ofabout 5 times of its wild type counterparts (FIG. 17B). Likewise,reprogramming CD3^(neg) T cell was about 5 times more efficient than thewild type counterparts (FIG. 18B). p53 deficient cell lines have beenreported be easier to reprogram since the apoptosis pathway is hindered.FAS knock-out further induces apoptosis resistance. While loss of TCRexpression makes T cells less healthy, an indication that apoptosisplays an important role in the process of reprogramming.

Example 9: Knockdown of TCR in T Cells with siRNA

FIG. 19 is a graph showing IFN-gamma production of wild type NY-ESO-1TCR (wt) or modified NY-ESO-1 TCR with a second disulfide bond andde-N-glycosylation to the beta chain (S/SD). RNA was electroporated intoT cells with endogenous T cell receptors (TCRs) knocked down with siRNA.IFN-gamma was detected by ELISA after the T cells were stimulated with aHLA-A2 positive cell line pulsed with NY-ESO-1 specific peptide,p156-165, for 18 h.

FIG. 20, comprising FIGS. 20A and 20B, shows TCR alpha knockdown by CAS9RNA and gRNA co-electroporation. Six days after electroporation, cellswere analyzed for TCR expression by assessing CD3.

FIG. 21 shows Sanger sequencing. Results show multiple peaks in CD3negative enriched T cells, with either CAS9 mRNA and gRNAselectroporated to knockdown TCR alpha (TRAC-5) or TCR beta (TRBC-7).

FIG. 22 is a panel of graphs showing CD3 negative T cells withendogenous TCR beta (TRB-7) knockdown re-expressed CD3 four hours afterNY-ESO-1 TCR alpha and beta (1G4LY95 TCR) RNA electroporation. Normal Tcells (ND424 Beads) were used as control, which showed nearly 100% CD3positive with 5.25% endogenous TCR vb13.1 expression.

FIG. 23, comprising FIGS. 23A-23D, is a panel of graphs showing knockdown of endogenous TCR enhanced with both transgene expression andfunction of TCR RNA electroporated T cells. FIG. 23A shows TCRexpression of T cells electroporated with TCR siRNA (solid openhistogram), control siRNA (dotted open histogram) and T cells withoutany siRNA (filled histogram). FIG. 23B shows transgene (TCR vb13.1)expression of wild type NY-ESO-1 TCR (wt) or modified TCR (SD) RNAelectroporated T cells with TCR siRNA, control siRNA, or no siRNA. FIG.23C shows NY-ESO-1 tetramer staining of wild type NY-ESO-1 TCR (wt) ormodified TCR (SD) RNA electroporated T cells with TCR siRNA, controlsiRNA, or no siRNA. FIG. 23D shows specific lysis of a HLA-A2/NY-ESO-1positive tumor line by TCR siRNA knockdown, wildtype NY-ESO-1 TCR RNAelectroporated T cells.

FIG. 24 is a graph showing fluorescence of tumor cells after injectionof T cells into a mouse model. Ten million Nalm6-CBG-ESO-GFP (clickbeetle green) tumor cells that expressed both NY-ESO-1 and GFP wereintravenously injected into NOD/SCID mice. Five days after tumorinoculation, CBR (click beetle red) transduced and RNA electroporated Tcells were injected as indicated in the different groups and tumorgrowth was monitored by bioluminescent image (BLI).

FIG. 25 show bioluminescent images of the mice from two groups that hadbeen treated by CD19BBZ CAR RNA T cells or modified NY-ESO-1 TCR RNA atdifferent time points.

Example 10: Universal CAR19 T Cells Generated by Combination ofLentiviral Transduction and Disruption of the TCR-CD3 Complex on T CellsUsing CRISPR

As shown in FIG. 26, primary T cell were stimulated withanti-CD3/anti-CD28 beads at day 0, and then transduced with lenti-CAR19.Over 70% of the cells were CAR19 positive as detected by flow cytometry.Since transient expression of CRISPR is sufficient to mediate geneknockout, a “hit-and-run” delivery strategy was developed to transientlyexpress CRISPR by utilizing electro-transfer of in vitro transcribed RNAencoding CAS9 and gRNAs targeting the constant regions of TCR α chain,TCR β chain, and beta-2 microglobulin gene on day 3. T cells werecultured for 24 hours at 32° C. after electrotransfer, then returned tonormal condition.

To measure TCR expression, a monoclonal antibody specific for CD3 wasused. CD3 was chosen as CD3 is only present on the cell surface whenTCRs are expressed. CRISPR constructs were electroporated into primary Tcells (FIG. 26). TCR single negative and TCR/HLA-A double negative cellswere expanded by exposure to CD19 presenting K562 cells, which resultedin >100 fold expansion (FIG. 27).

After expansion, the cells remained TCR single negative or TCR/HLA-Adouble negative, and the CAR19 positive population was enriched.Endogenous TCR expression remained negative in TCR single negativecells, while TCR and HLA-A expression remained negative in TCR/HLA-Adouble negative T cells after K562-CD19 stimulated expansion (FIG. 28A).CAR19 positive cells were enriched by K562-CD19 stimulated expansion(FIG. 28B).

The majority of expanded universal T cells were CD45RO positive (FIG.29A) and retained high levels of CD62L expression (FIG. 29B), mediumlevels of CD28 expression (FIG. 29A) and low levels of CCR7 expression(FIG. 29B).

CRISPR gene editing did not affect the anti-tumor activity of universalCAR19 T cells in vitro (FIG. 30A). Depletion of TCR or TCR/HLA-A hadminimal effect on CAR19 expression and anti-tumor activity (FIGS. 30Band 30C). TCR single and TCR/HLA-A double negative CAR19 T showed robustlytic capacity when challenged with Nalm6 tumor cells (FIG. 30B). CD107arelease and cytokine secretion also showed potent anti-tumor activity inthe universal cells (FIG. 30C). TCR single ablation or TCR and HLA-Adouble ablation CAR19 T cells exhibited similar proliferation kineticsafter challenge with CD19 expressing cells (FIG. 30D).

To test the anti-tumor activity of CRISPR/CAS9 edited CAR19 T cells, TCRsingle negative, TCR and HLA-A double negative CAR19 T were infused intoNSG mice bearing Nalm6 tumor cells. All the mice receiving unmanipulatedT cells and mice infused with lentiviral GFP transduced wild type Tcells died within 3 weeks after tumor cell infusion. Objective tumorregression was observed for mice receiving CAR19 T cells (FIG. 6).CRISPR/CAS9 was found to not affect the in vivo tumor killing activityof CAR19 T cells, thus, confirming the advantage of combining lentiviralgene transfer and CRISPR/CAS9 for T cell therapy.

Full ablation of TCR α and β chains and HLA-A molecule on T cellscompletely abrogated non-specific killing when the cells were challengedwith HLA unmatched tumor cell lines (FIG. 32A). Elimination of HLA-Amolecules activated NK cells after a long period of co-culture (5 days).No off-target activity was observed when these cells were challenged byallogeneic whole blood PBMC after 24 hours in an IFNr Elispot assay. Thelack of off-target activity suggests T cells may play a dominant role inacute immune responses after encountering allogeneic cells. All of theresults suggest that CRISPR/CAS9 edited TCR α and β chains and HLA-Amolecules (triple negative) T cells could serve as a source of universaleffector donor cells.

CAS9 and different gRNAs targeting FAS were electro-transferred into Tcells. FASneg cells were sorted and then transduced with lentiviralCAR19. Flow cytometry and Sanger sequencing data showed that FAS hadbeen modified by the CRISPRs (FIG. 33). CAR19 gene expression of FASnegT cells was comparable to the wild type. Even after a short period ofincubation with Nalm6 tumor cells, CD107a expression was greatlyenhanced in FASneg CAR19 T cells compared to wild type counterpart cellseven within 4 hours of co-culture.

Some reports showed that even weak antigenic stimuli can trigger FASactivation to promote T cell proliferation (Rethi, et al, Blood, vol.112(4):1195-1204, 2008). Interestingly, FASneg CAR19 T cells expandedmuch quicker than the wild type CAR19 T cells when the cells werestimulated by high levels of CD19+K562 cells. This suggests thatFAS/FASL triggered apoptosis instead of activation under high levelantigenic conditions (FIG. 34A). FASneg CAR19 T cells further showedreduced apoptosis levels as measured by Annexin V staining (FIG. 34B).

As had been observed in vitro, FASneg T cell showed enhancedproliferation as compared to wild type T cells. Similar proliferationresults were observed when a True Count assay of CAR19 T cells wasperformed after infusion of the cells into Nalm6 bearing mice. TheFASneg CAR19 group showed superior anti-tumor activity when compared tothe wild type group (FIG. 35B). This difference is illustrated in thegraph of FIG. 35C showing the bioilluminescence data between those twogroups. These data indicate that FAS ablation in CART cells enhanced itsanti-tumor activity.

CAS9 and different gRNAs targeting PD1 were electro-transferred into Tcells after lentiviral transduction with PSCA-CAR. PD1 knock out cellswere confirmed by surface PD1 expression after CD3/CD28 bead stimulation(FIG. 36). PD1 negative cells were enriched by microbead depletion andthen stimulated with PSCA antigen presenting PC3 tumor cells. PSCA-CARpositive cells were enriched both in the wild type and the PD1 negativegroups. After incubation with PC3-PSCA-PDL1 tumor cells, PD1 expressionwas quickly upregulated on the surface of wild type PSCA-CAR T cells,with very low levels of PD1 expression detected on PD1 negative PSCA-CART cells (FIG. 37). PD1 negative PSCA-CAR T cells also showed greatlyenhanced and sustained high levels of CD137 expression (FIG. 37), amarker of T cell activation, indicating that the PD1/PDL1 inhibitorysignaling pathway was blocked.

When tested in an in vivo PC3-PSCA-PDL1 NSG model, significant enhancedanti-tumor activity was detected in the PD1 negative PSCA-CAR T cellgroup compared to the wild type group (FIGS. 38A and 38B) suggesting atherapeutic value of PD1 ablation for CART cell therapy.

To test the graft vs host disease (GVHD) effect of CRISPR engineereduniversal CART cells, a high T cell dose was given to NSG mice withNalm6 leukemia. The mice were treated with double or triple knock outCART cells and did not show any signs of developing GVHD. By contrast, 3out of 4 mice from the wild-type CD19 CART group developed GVHD by day65, which was confirmed by histological examination of different organs(FIG. 39).

In another experiment, the cells were resuspended in FBS and infusedintravenously into mice after a sub-lethal irradiation. Clinical GVHDwas monitored 2 to 3 times per week. Four out 5 mice receiving wild typeT cells died during the 60 day study, while PBS treated, TCR single andTCR/HLA-I double ablated T cell treated groups did not show any signs ofGVHD. Mice receiving wild type T cells underwent body weight loss.However, PBS treated, TCR single and TCR/HLA-I double ablated T celltreated groups slightly gained weight during the study (FIGS. 40A and40B).

T cells were treated with Cas9 and gRNAs targeting CD3, B2M and PD1 orFas after lentiviral CD19-CAR transduction. Triple knock out universalCART cells were injected into mice bearing Nalm6-PDL1 tumors. Superioranti-tumor activity was observed in mice receiving PD1/CD3/HLA-I tripleknock out cells as compared to CD3/HLA-I double knock out cells, furtherindicating the therapeutic value of blocking the PD1 signaling pathway(FIGS. 41A and 41B). These data supply a way to enhance the treatment ofuniversal CART cells with CRISPR/Cas9.

As gRNAs are prone to degrade, a simplified one-shot method wasdeveloped to generate universal CART cells. gRNAs were constitutivelyexpressed together with CARs in a single lentiviral vector. Naïve Tcells were transduced by lentivirus encoding gRNAs and CARs one dayafter stimulation with CD3/CD28 Dynabeads. The cells were electroporatedwith Cas9 mRNA at day 3 (FIG. 42). This system allows the manipulationof several genes with one vector (FIG. 42). CD3 expression was confirmedby flow cytometry at day 6. T cells treated with the one-shot systemshowed consistent gene ablation as high as 90% in each of the differentCas9 mRNA groups (FIG. 43).

Progress in adoptive T-cell therapy for cancer and infectious diseaseshas been hampered by the lack of readily available antigen-specifichuman T lymphocytes. Pluripotent stem cells could provide an unlimitedsource of T lymphocytes. To address this issue, expression of FAS, PD1,CTLA4, and PPP2r2d was disrupted in primary cells and T cells.

Sendai virus was used to reprogram primary cells and T cells. There aremultiple methods available for the generation of iPSCs, includingvirus-mediated gene transduction and chemical induction. Whilelentiviral and retroviral vectors require integration into hostchromosomes to express reprogramming genes, DNA-based vectors, such asadenovirus, adeno-associated virus, and plasmid vectors, existepisomally and do not require integration, however, they may still beintegrated into host chromosomes at certain frequencies, and thereprogramming efficiency is relatively low. Likewise, mRNA basedreprogramming is complicated and has proven to be extremely inefficient.

In contrast, Sendai virus does not integrate into the host genome oralter the genetic information of the host cell. Sendai virus also hasreprogramming potential comparable to lentiviral- and retroviral-basedgene transduction.

Each well in a 24 well plate was seeded with 0.1 million wild type,FASneg, CD3neg TCR alpha and beta chain knock-out T cells. The cellswere stimulated with CD3/CD28 beads. At day 3 post stimulation, thebeads were removed, the cells were resuspended in 1 mL of pre-warmed Tcell complete medium, and then incubated with a calculated volume ofCytoTune Sendai virus comprising a polycistronic vector for expressionof hKlf4, hOct3/4 and hSox2 in the cells (Lifetechnologies, Carlsbad,Calif.). Treated T cells were seeded in 24 well plates, and centrifugedat 2250 rpm for 90 minutes at room temperature. An additional 1 mL ofcomplete T cell medium was added to each well and the plate wasincubated overnight at 37° C. in a humidified atmosphere of 5% CO2.

On the day after transduction, Sendai virus was removed by washing the Tcells with fresh complete medium and culturing the cells for 2 days.Media was half changed every day. On day 3 after infection, cells weretransferred to MEF feeder plates and cultured in T cell medium withoutany cytokines. Four days after infection, the cells were cultured instandard hES medium. Media was changed every day. ES-like colonies wereobserved around day 7. The cells were cultured in conditioned hES mediumfrom day 15 and cultures continued for an additional 10 days. Colonieswere picked at around 25 to 30 days after transduction.

Around day 4, cell clumps were formed on feeder cells, indicating theinitiation of the reprogramming process. T cells went through dramaticmorphological changes during the reprogramming process to iPSCs (FIG.44A). Around day 12, large cell clumps with loose edges began to emerge.Around day 18, T cells were transformed to typical ES-like colonies withwell-defined edges. FASneg T cells were reprogrammed to iPSCs at anefficiency of about 5 times of the wild type counterparts (FIG. 44B).p53 deficient cell lines have been reported as easier to reprogram dueto the hindrance of the apoptosis pathway. FAS knock out may facilitatethe reprogramming process using a similar mechanism.

ES-like morphology of iPSCs reprogrammed from CD3neg TCR alpha or betachain knock out T cells was observed (FIG. 45A). The morphology remainedconstant after several passages. Reprogramming of CD3neg T cells wasabout 5 times less efficient than the wild type counterparts (FIG. 45B),suggesting that TCR knock-out may play a role in the process of T cellreprogramming or affect the cell viability after Sendai virus infection.FIG. 45C is a panel of images showing phosphatase staining of CD3negiPSC cells.

Typical embryonic stem cell morphology was observed indicating that theFASneg, CD3neg TCR alpha and beta chain knock-out T cells were inducedto a pluripotent state under defined reprogramming conditions. Whileloss of TCR expression makes T cells less healthy, the data describedherein suggests that apoptosis plays an important role in the process ofreprogramming.

Induction of endogenous pluripotent stem cell genes was also detected inthe different T-iPSC cell lines (FIG. 46). Immunostainings for Tra-1-60and SSEA4 expression further indicated the stem cell phenotype of theT-iPSC cells (FIG. 47A). Fas knock out was confirmed in the T-iPSCs bySanger sequencing (FIG. 47B).

dCas9 and FokI-Cas9 were reported to have less off-target activity. Tcells were evaluated if they could be edited by a modified version ofthe CRISPR/dCAS9 and CRISPR/FokI-CAS9 system (FIG. 48A). Flow cytometricdata showed primary T cells were edited by both CRISPR/dCAS9 andCRISPR/FokI-CAS9 (FIG. 48B). The CRISPR/dCAS9 gene knock out systemexhibited enhanced specificity with at least one pair of gRNAs,rendering the knock out event more precise and more specific.

To test the off-target events of CRISPR/CAS9 in T cells, a surveyorassay was performed at off target sites. For the genes tested, noobvious cleavage was observed at the genomic loci (FIG. 48C).

Example 11: Multiplex Genome Editing

CART cells were generated by using a CRISPR/Cas9 system tosimultaneously disrupt multiple genomic loci. The CART cells weredeficient in the expression of endogenous TCR and HLA class I (HLA-I)molecules for use as allogeneic universal CART cells. T cell receptor(TCR) α chain, TCR β chain and beta-2 microglobulin (B2M) genes weredisrupted with high efficiency through the co-electroporation of mRNAencoding Cas9 with gRNAs targeting these genes. Universal TCR or CARTcells were generated by combining lentiviral (LV) delivery of CAR andCRISPR RNA electroporation to disrupt endogenous TCR and B2M genessimultaneously. In addition, disruption of endogenous PD1 enhanced theefficacy of CAR therapy in a solid tumor model.

Multiple Deliveries of gRNAs Disrupts Genes in Human Primary T Cellswith High Efficiency without Impairing Effector Function

Efficient multiplex genomic editing is required to generate universal Tcells that are deficient in TCR, HLA and other genes. CRISPR/gRNA RNAelectroporation was optimized to achieve efficient gene disruption in Tcells. First, Cas9 and gRNAs were co-electroporated with RNA generatedusing an in vitro transcription system (FIG. 49, left), and a“hit-and-run” delivery strategy was developed to transiently deliver theCas9 mRNA and gRNAs to T cells by electroporation (FIG. 49, right).

An initial experiment targeting the TCR β constant region (TRAC) or □constant region (TRBC) with single electroporation resulted in 1% to 3%CD3-negative (CD3^(neg)) T cells, respectively, (FIG. 50A, uppergraphs). To determine if transient exposure to mild hypothermia allowedmore efficient gene disruption, cells were edited at 37° C. or 32° C.CRISPR-mediated disruption of TRAC and TRB was increased up to 4-foldwhen T cells were cultured for 24 h at 32° C. after Cas9/gRNAco-electroporation (FIG. 50A, lower graphs). The optimal molecular ratioof Cas9:gRNA for maximum disruption efficiency was 1:1 to 2:1, and thegene disruption efficiency was correlated with the amount ofelectro-transferred mRNA (FIG. 51A).

Compared with mRNA, gRNAs are more prone to rapid degradation, whichpotentially limits the targeting efficiency. Thus, multiple, sequentialelectroporations of gRNA were tested after the initial Cas9/gRNAelectroporation. There was a marked increase in disruption frequency atthe protein level, as 82.4% of cells were CD3^(neg) after the third gRNAelectroporation (FIG. 50B). Clonal sequencing showed that the genomictargeting efficiency reached 89.4% after the third gRNA electroporation(FIG. 51B). A surveyor assay confirmed a cleavage rate of 81.7% and49.3% at the genomic loci of TRAC and TRBC, respectively, after a thirdelectroporation of gRNAs (FIG. 52). Multiple peaks in the Sangersequencing data flanking the TRAC and TRBC target sites confirmed thatthe genomic reading frame shifted downstream of the target sites (FIG.53A). The occurrence of insertions or deletions (indels) caused by theNHEJ mediated by CRISPR/Cas9 was confirmed by clonal sequencing (FIG.53B). The TCR disrupted TCR/CD3^(neg) population was enriched to over99% (99.70±0.20%) by a single step of CD3 negative selection (FIG. 54).

To develop methods to expand the TCR/CD3^(neg) T cells, TCR/CD3^(neg) Tcells were co-electroporated with the HLA-A2 restricted 1G4 NY-ESO-1 TCR(□□□) RNAs to restore CD3 expression (FIG. 55, left panel). Following Tcell stimulation/expansion methods, the following were compared: 1) arapid T cell expansion protocol (REP) using PBMC as feeder cells, 2)anti-CD3/CD28 Dynabeads (Beads), or 3) OKT3 loaded K562-based artificialantigen-presenting cells expressing ligands for CD28 and 4-1BB (K562aAPC). TCR/CD3^(neg) T cells were also electroporated with CD19 CAR RNA(FIG. 55, right panel) and then stimulated by irradiated K562 aAPC thatexpressed CD19 (K562-CD19). Fold expansion values of 751.0±217.1,35.7±9.3, 46.3±8.5 and 57.5±5.0 were achieved for REP, Beads, K562 aAPCand K562-CD19, respectively, after a single stimulation for 10 days(FIG. 56).

To test whether CRISPR/Cas9 gene editing would affect the phenotype andfunction of the T cells, the phenotype of TCR/CD3^(neg) T cells expandedby the different methods was examined and showed that all of theexpanded cells remained CD3 negative and most retained a high level ofCD27 (from 79.8% to 93.4%), consistent with a central memory cellphenotype (FIG. 57). The expanded TCR/CD3^(neg) T cells wereelectroporated a second time with CD19 CAR mRNA to test their anti-tumoractivities. The surface CAR expression of the TCR/CD3^(neg) T cells wasequal to that of the control group (FIG. 58). When the TCR/CD3^(neg)CD19 CAR T cells were stimulated with CD19⁺ Nalm6 leukemia cells, theCD107a up-regulation (FIG. 59A), cytokine secretion (FIG. 59C) andkilling activity (FIG. 59B) of CD19 CAR⁺ TCR/CD3^(neg) T cells wasequivalent to those of the wild-type control cells. The CD19 CARTCR/CD3^(neg) T cells were infused into Nalm6-bearing NSG mice to testtheir in vivo anti-tumor activity. Tumor regression was evident with anefficacy equivalent to that for the CART19 wild-type counterpart cells(FIGS. 59D and 59E). The results indicate that CRISPR/Cas9 editing ofthe endogenous TCR did not adversely affect the function of primary Tcells for adoptive immunotherapy.

Reduced alloreactivity of TCR α, β and B2M triple-disrupted T cells.

Disrupting both TCR α and β chains is required to prevent TCRmiss-pairing-associated toxicity for TCR-redirected T cell adoptiveimmunotherapy and B2M is essential for the assembly and expression ofHLA-I complex. In view of this, TCR α and β chains and B2M tripledisruption was developed to generate universal T cells. First, theability of eliminating HLA-I expression on the T cells by disrupting B2Mwas tested. T cells were electroporated with B2M-targeting Cas9/gRNARNA. This resulted in a B2M and HLA-I double-negative population of79.9%. The HLA-I^(neg) population could be further enriched by negativeselection (FIG. 60).

To generate triple-knockout T cells lacking the TCR α, β chains and B2M,Cas9 mRNA was co-electroporated with three different gRNAs targetingTRAC, TRBC and B2M. As a result, the CD3 and HLA-I double-negative cellpopulation was 65.4% (FIG. 61). After enrichment of the double andtriple knockout cells, the TCR α and β chains and B2M triple-knockout Tcells abrogated the non-specific killing of HLA unmatched tumor celllines (FIG. 62). No response was observed when these cells werechallenged by allogeneic whole-blood irradiated PBMCs in an IFN□ Elispotassay (FIG. 63, left panel). The ablation of HLA-I molecules alsosharply reduced the allo-reactivity, as confirmed by co-culture ofallogenic PBMCs with irradiated B2M-disrupted cells (FIG. 63, rightpanel). The results above suggest that triple-negative T cells that lackTCR α and β chains and B2M could potentially serve as a source ofuniversal T cells for adoptive immunotherapy, resisting rejection by thehost immune system while unable to cause graft versus host disease.

Improved Anti-Tumor Activity of TCR Redirected, Endogenous TCR-DisruptedT Cells.

T cells with CRISPR/Cas9-disruption of TCR α and β chains showedelevated transgenic TCR expression on the cell surface after beingredirected with an NY-ESO-1 TCR (1G4). Transgenic TCR expression was67.6%, 78.8% or 94.3% for the TCR □ or □□□□ chain single knockout or the□□□□ double knockout, respectively, compared with 46.8% for wild-type Tcells. The improved transgenic TCR expression led to enhanced T cellfunction, as evidenced by increased antigen-specific CD107a expression(FIG. 65A) and enhanced cytotoxicity (FIG. 65B), especially for the □□□double-knockout T cells.

In a separate experiment, □□□ double-knockout T cells were transfectedwith a different NY-ESO-1 TCR (8F). Relative to 1G4 TCR, this 8F TCRexhibited a higher significant improvements in both transgenic TCRexpression (FIG. 66; 60.1% for TCR/CD3^(neg) versus 44.7% (with ˜5%endogenous TCR Vβ8 background) for wild-type T cells (Cas9 Mock Tcells)) and function (CD107a expression in FIG. 67A, and cytokineproduction in FIG. 67B). These results highlight the differentialinfluence of endogenous TCR on transgenic TCR expression and function.

Universal CART Cells Retain Antitumor Efficacy and do not Cause GVHD.

Universal CD19 CART cells were generated by combining LV transduction ofCD19 CAR with RNA electroporation of Cas9/gRNAs (FIG. 68). The cellswere expanded and the remaining CD3^(neg) cells had high levels of CD19CAR expression (FIG. 69). The majority of the expanded T cells wereCD45RO positive and retained a high level of CD62L expression and amedium level of CD28 expression, consistent with a central memory cellstatus (FIG. 70). The expanded TCR/HLA-I double-negative CD19 CARTshowed robust in vitro anti-tumor activities, such as CD107a release(FIG. 71), cytokine secretion (FIG. 72), lytic capacity (FIG. 73), andproliferation (FIG. 74), that was as potent as those of the wild-typeCD19 CART cells.

The T cells were infused into NSG mice bearing disseminated Nalm6leukemia. Mice treated with CART cells with a disrupted endogenous TCR(LV-CD19 CAR TCR's) or with a simultaneous disruption of TCR and HLA-I(LV-CD19 CAR TCR/HLA-I^(neg)) exhibited tumor regression similar to thatof mice treated with wild-type CD19 CART cells (LV-CD19 CAR) (FIGS. 75Aand 75B), suggesting that the disruption of TCR alone or together withB2M did not affect CART cell anti-tumor activity.

To test the GVHD effect of the engineered T cells, a high T cell dose(20×10⁶/mouse) was given to NSG mice with Nalm6 leukemia. As shown inFIG. 76, mice treated with CD19 CART cells with TCR disruption alone(LV-CD19 CAR TCR/CD3^(neg)) or the simultaneous disruption of TCR andB2M (LV-CD19 CAR TCR/HLA-I^(neg)) exhibited similar tumor regressioncompared with that of the wild-type CD19 CAR T cells (LV-CD19 CAR). Micetreated with the double or triple knock out CAR T cells did not developany signs of GVHD. By contrast, 3 out of 4 mice from the wild-type CD19CART (LV-CD19 CAR) group developed GVHD at day 65, which was confirmedby histological examination of different organs. Thus, the disruption ofTCR alone or together with HLA-I did not affect the in vivo anti-tumoractivity of CART cells while eliminating alloreactivity.

Adenoviral CRISPR Delivery into Primary T Cells.

The CRISPR/Cas9 system are rapidly being harnessed for gene regulationand gene editing purposes in model organisms and cell lines. Viralvectors may be particularly fit to broaden the applicability of CRISPRto other cell types, including dividing and quiescent primary cells.Adenovirus, namely second-generation fiber-modified adenovirus encodingCas9 and single guide RNA (gRNA) molecules, were used to bring Cas9nuclease to the PD1, Fas and TRAC loci (FIG. 77). Adenoviral-mediatedtransduction of CRISPR into tumor cells (FIG. 78) yielded high rates oftargeted mutagenesis of up to about 71% (FIGS. 79A and 79B). Adenovirusappears to constitute a valuable platform for introducing CRISPR intohuman T cells regardless of their quiescent status. This approach willaid investigating the potential for CRISPR gene regulation and editingin numerous experimental settings.

Electroporation Optimization.

CD3 and B2M knock-out efficiency and T cell expansion were assessedafter Cas9 and gRNA electroporation (EP) in 4 mm cuvettes and 2 mmcuvettes. Standard EP conditions with a 2 mm cuvette (360 v/1 ms, 1^(st)EP-20 μg Cas9 RNA+10 μg gRNA/100 μl T cells, 2^(nd) EP 5 μg gRNA/100 μlT cells) showed the highest CD3 and B2M knockout percentages, 81.8% and88.6%, respectively, with T cell expansion at about 2.7 fold (EP #1),compared with about 18.8 fold expansion of control EP T cells (EP #12).Decreasing the gRNA dose (EP #2-5) dramatically increased T cellexpansion, but only slightly affected CD3 and B2M knock-out efficiency.See FIG. 80. Standard EP conditions with a 4 mm cuvette resulted indramatically decreased CD3 and B2M knockout efficiency (EP #8),suggesting that the EP conditions (voltage or/and pulse length) need tobe further optimized for use with 4 mm cuvettes.

Compared with standard electroporation (EP) conditions in a 2 mm cuvette(EP #10-13) or 4 mm cuvette. High CD3/B2M knockout efficiency wasobserved with improved T cell fold expansion (EP #1 and 5). See FIG. 81.

To further optimize EP conditions to achieve maximum T cell foldexpansion with CD3/B2M knockout efficiency over 60%, different EPconditions and RNA amounts were tested. The results showed that foldexpansion was improved with relatively high CD3/B2M knockout efficiency(63.5% for CD3 and 84.8% for B2M) for EP #4 (400 v/2 ms/120 μg CAS9 RNA)for EP #1 and (500 v/1 ms/20 μg gRNA) for EP #2. See FIG. 82.

Additional experiments were performed to optimize EP conditions. Resultsshowed that compared with the most favorable condition tested (EP #1 inFIG. 82), using 500 v/1 ms/120 μg CAS9 RNA (EP #1) and 500 v/1 ms/20 μggRNA (EP #2) produced increased CD3/B2M knockout efficiency and T cellexpansion (EP #3). See FIG. 83.

Large-Scale Electroporation and Expansion.

Experiments were performed to determine if large-scale electroporationscould yield high knock-out and expansion efficiencies. On day 0,anti-CD3/anti-CD28 beads were used to stimulate T cells obtained from 3donors (100×10⁶ cells/donor, concentrated to 0.5×10⁶/ml). On day 1,stimulated T cells were transduced with CD19 CAR lentivirus. 50 mL(25×10⁶ cells) of T cells were reserved as unmodified T cells (Group 9).On Day 3, the beads were removed and the transduced T cells from eachdonor were separated into two groups, CART/mock EP (10 mL, 5×10⁶) andCART/CRISPR (10 mL, 50×10⁶). The cells were then electroporated withCAS9 RNA (1st EP) and Groups 1, 3, 5 and 7 cells were split. On day 4,the gRNA was electroporated into the T cells and the cells were culturedat 1×10⁶ cells/mL. On days 5 and 7, the cells were split. On day 8, CD3+cells were removed from Groups 2, 4 and 6. On day 11, the T cells wereharvested and 25×10³ cells from the three donors were sent forkaryotyping.

TABLE 1 Experimental groups. Group # Donor T cells 1 ND391 CART/MOCK EP2 ND391 CART/CRISPR 3 ND463 CART/MOCK EP 4 ND463 CART/CRISPR 5 ND463UNMOD 6 ND469 CART/MOCK EP 7 ND469 CART/CRISPR

T cell numbers (upper chart of FIG. 85) and fold expansion (lower chartof FIG. 85) were assessed after the electroporation and culturingprocedure. Fold expansion of the T cells transduced with CD19 CAR alone(TD alone) or transduced with CD19 CAR and edited with CRISPR (TD/KO) isshown in the left graph of FIG. 86 and fold expansion of the T cells onday 10 is shown in the right graph of FIG. 86. By optimizingelectroporation conditions and CAS9/gRNA doses, approx. 60-70% CD3/B2Mknock-down efficiency and approx. 30 fold T cell expansion was observedafter 10 days (FIG. 87 shows CD3/B2M/CAR expression at day 10).

Eight days after CD3/CD28 bead stimulation and CRISPR RNAelectroporation, CD3 positive T cells were removed. FIG. 88 showsCD3/B2M expression in the three donor populations at day 8. On day 11, Tcells were subjected to FACS staining to detect CD3, B2M and CARexpression. ND463 non-transduced (NOTD) were used as a negative control.FIG. 89 shows CD3 and B2M expression in CD19 CAR TD (transduced)/CRISPRelectroporated, CD3 depleted T cells; CD19 CAR TD/CRISPR electroporatedT cells; and CD19 CAR TD T cells. FIG. 90 shows CAR expression in CD19CAR TD/CRISPR electroporated, CD3 depleted T cells; CD19 CAR TD/CRISPRelectroporated T cells; and CD19 CAR TD T cells. FIG. 91 showsCD3/B2M/CAR expression on day 11 in CD19 CAR TD (transduced)/CRISPRelectroporated, CD3 depleted T cells; CD19 CAR TD/CRISPR electroporatedT cells; and CD19 CAR TD T cells. FIG. 92 summarizes CD3/B2M/CARexpression in the different T cells groups.

On day 11 the different T cell groups, as indicated in FIG. 93, werestimulated by a CD19 positive cell lines, Raji or Nalm6. K562 was usedas a CD19 negative control. After 4 hr of coculturing, CD107aup-regulation was detected in each of the T cell groups, except thenegative controls.

On day 11, killing ability of the T cells, as indicated in FIG. 94, weretested using a luminescent cytotoxic lymphocyte (CTL) assay aftercoculturing the T cells with CD19 positive target cells, Nalm6-CBG. Alsoon day 11, cytokine production of the T cells was analyzed bystimulating the T cell groups with Nalm6 target cells, see FIG. 95.

The T cells were cultured in medium containing 100 U/ml of IL-2 for upto 26 days. The results shown in FIG. 96 indicate no abnormal T cellgrowth was observed for the CRISPR edited T cells from the three donors.

As one of most attractive applications of the CRISPR/Cas9 system,multiplex genome editing holds great promise for advancing T cell-basedadoptive immunotherapy. However, the low targeting efficiency of DNAtransfection limits the use of multiplex genome engineering in primary Tcells. A “hit-and-run” delivery strategy was developed to introduceCRISPRs to T cells via the co-electroporation of Cas9 mRNA and gRNA.Through a combination of up to three rounds of gRNA electroporation withtransient exposure to mild hypothermia, a targeting efficiency of >80%at the protein level was routinely achieved for a single genedisruption. More encouragingly, triple gene disruption of TRAC, TRBC andB2M yielded double negative CD3 and HLA-I at about 65% without anypurification and selection. The results also demonstrate that enrichmentto >99% purity of gene-disrupted T cells was easily achieved usingclinically approved paramagnetic beads and that the purified T cellswere expanded up to 500-fold in 10 days. The expanded T cells maintainedtheir gene disruption phenotype and displayed features consistent withcentral memory T cells. The disrupted T cells did not cause GVHD,suggesting that they may be used as allogeneic CAR T cells. Importantly,the gene-edited T cells showed anti-tumor activities both in vitro andin different tumor mouse models that were as potent or more potent thannon-gene edited T cells. Thus, the process described herein to generatesynthetic cells could be easily translated into current GMP-compliantmanufacturing procedures.

The data described herein demonstrates that CRISPR/Cas9 is a powerfulmultiplex genome editing tool in primary human T cells. Previous reportshave shown that T cells can be genetically edited by ZFNs or TALEN toeliminate the expression of the endogenous TCR α and β chains to avoidGVHD. Due to the complexity of the targeting strategies for manipulatingmultiple genes by zinc finger nucleases (ZFN) and TAL effector nucleaseTALEN in T cells, previous studies have not been able to prevent GVHDand host-versus-graft reaction simultaneously in pre-clinical animalmodels. NK cell activation can also be aborted by the ablation ofstimulatory NK ligands by CRISPR/Cas9 or by the expression ofnonclassical HLA class I molecules such as HLA-E, which couldpotentially protect universal T cells from NK-cell-mediated rejection.

In summary clinical scale universal CART cells, with potent anti-tumoractivity and reduced alloreactivity can be efficiently generated usingmultiplex CRISPR technology. This approach can be incorporated intocurrent GMP-compliant manufacturing procedures and has a high potentialfor translation, given the successful translation of adoptive transfertherapy with ZFNs for HIV/AIDS. It is possible that universal CAR andTCR T cells will provide an alternative to autologous T cells. Indeed,it is conceivable that universal CAR and TCR T cells with disabledcheckpoint molecules may be more efficacious and have wider use thancurrent CART therapy using autologous T cells against cancers andinfectious diseases.

OTHER EMBODIMENTS

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While this invention has been disclosed with referenceto specific embodiments, it is apparent that other embodiments andvariations of this invention may be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims are intended to be construed to include all suchembodiments and equivalent variations.

1-34. (canceled)
 35. The method of claim 36, wherein the condition isassociated with enhanced immunity.
 36. A method of treating a conditionin a subject, comprising administering to the subject a therapeuticallyeffective amount of a pharmaceutical composition comprising aCRISPR-modified T cell, wherein the CRISPR-modified T cell comprises:(a) a CRISPR-mediated insertion or deletion in an endogenous gene locuscausing the downregulation of the endogenous gene expression, whereinthe endogenous gene is selected from the group consisting of TCR α chain(TRAC), TCR β chain (TRBC), beta-2 microglobulin (B2M), a HLA molecule,CTLA-4, PD1, and FAS; and (b) a nucleic acid encoding a modified T cellreceptor (TCR) having an affinity for a surface antigen on a targetcell, wherein the modified TCR comprises a TCR alpha chain and TCR betachain.
 37. The method of claim 36, wherein the condition is anautoimmune disease.
 38. The method of claim 37, wherein the autoimmunedisease is selected from the group consisting of AcquiredImmunodeficiency Syndrome (AIDS), alopecia areata, ankylosingspondylitis, antiphospholipid syndrome, autoimmune Addison's disease,autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner eardisease (AIED), autoimmune lymphoproliferative syndrome (ALPS),autoimmune thrombocytopenic purpura (ATP), Behcet's disease,cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigueimmune dysfunction syndrome (CFIDS), chronic inflammatory demyelinatingpolyneuropathy (CIPD), cicatricial pemphigold, cold agglutinin disease,crest syndrome, Crohn's disease, Degos' disease,dermatomyositis-juvenile, discoid lupus, essential mixedcryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease,Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonaryfibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy,insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still'sdisease), juvenile rheumatoid arthritis, Meniere's disease, mixedconnective tissue disease, multiple sclerosis, myasthenia gravis,pernacious anemia, polyarteritis nodosa, polychondritis, polyglandularsyndromes, polymyalgia rheumatica, polymyositis and dermatomyositis,primary agammaglobulinemia, primary biliary cirrhosis, psoriasis,psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumaticfever, rheumatoid arthritis, sarcoidosis, scleroderma (progressivesystemic sclerosis (PSS), also known as systemic sclerosis (SS)),Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus,Takayasu arteritis, temporal arteritis/giant cell arteritis, ulcerativecolitis, uveitis, vitiligo, Wegener's granulomatosis, and anycombination thereof.
 39. The method of claim 36, wherein the conditionis a cancer.
 40. The method of claim 39, wherein the cancer is selectedfrom the group consisting of breast cancer, prostate cancer, ovariancancer, cervical cancer, skin cancer, pancreatic cancer, colorectalcancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia,lung cancer, and any combination thereof. 41-47. (canceled)
 48. Themethod of claim 36, wherein the modified TCR is selected from the groupconsisting of a wild-type TCR, a high affinity TCR, and a chimeric TCR.49. The method of claim 36, wherein the modified TCR comprises: (a) atleast one disulfide bond; (b) a co-stimulatory signaling domain at a C′terminal of at least one of the chains; or (c) at least one murineconstant region.
 50. The method of claim 49, wherein the co-stimulatorysignaling domain is (i) selected from CD27, CD28, 4-1BB, OX40, CD30,CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2,CD7, LIGHT, NKG2C, or B7-H3 co-stimulatory signaling domain; or (ii) a4-1BB co-stimulatory signaling domain.
 51. The method of claim 36,wherein the TCR beta chain or the TCR alpha chain comprises at least oneN-deglycosylation.
 52. The method of claim 36, wherein the modified TCRhas higher affinity for the target cell a surface antigen when comparedwith the affinity of a wild-type TCR for the target surface antigen. 53.The method of claim 36, wherein the modified TCR is an 8F TCR.
 54. Themethod of claim 36, wherein the target cell surface antigen is selectedfrom the group consisting of viral antigen, bacterial antigen, parasiticantigen, tumor cell associated antigen (TAA), disease cell associatedantigen, and any fragment thereof.
 55. The method of claim 36, whereinthe target cell surface antigen is a tumor cell associated antigen(TAA), or an NY-ESO-1 tumor associated antigen.
 56. The method of claim36, further comprising an exogenous nucleic acid encoding acostimulatory molecule, wherein the co-stimulatory molecule is selectedfrom the group consisting of CD3, CD27, CD28, CD83, CD86, CD127, 4-1BB,4-1BBL, PD1 and PD1L.
 57. The method of claim 56, wherein the CD3comprises at least two different CD3 chains; or the CD3 comprises a CD3zeta chain and CD3 epsilon chain.
 58. The method of claim 36, wherein atleast one of the CRISPR-mediated insertions or deletions are in the TRACgene nucleotide sequence comprising SEQ ID NO: 1
 59. The method of claim36, wherein the CRISPR-modified T cell comprises a CRISPR-mediatedinsertion or deletion in the TCR α chain (TRAC) gene locus and the TCR βchain (TRBC) gene locus, causing the downregulation of the geneexpression of the TCR α chain (TRAC) gene and the TCR β chain (TRBC)gene.
 60. The method of claim 59, wherein the CRISPR-modified T cellfurther comprises a CRISPR-mediated insertion or deletion in: (a) thePD1 gene locus, causing the downregulation of the gene expression of theTCR α chain, the TCR β chain, and the PD1 gene; (b) the beta-2microglobulin (B2M) gene locus, causing the downregulation of the geneexpression of the B2M gene; or (c) the beta-2 microglobulin (B2M) genelocus, and the HLA molecule gene locus, causing the downregulation ofthe gene expression of the B2M, and the HLA molecule genes.
 61. A methodof treating a condition in a subject, comprising administering to thesubject a therapeutically effective amount of a pharmaceuticalcomposition comprising: (a) a CRISPR-modified T cell, wherein theCRISPR-modified T cell comprises a CRISPR-mediated insertion or deletionin an endogenous TCR α chain (TRAC) gene locus and an endogenous TCR βchain (TRBC) gene locus, causing the downregulation of the endogenousTCR α chain (TRAC) and TCR β chain (TRBC) genes expression, wherein atleast one of the insertions or deletions in the TRAC gene locus arewithin a TRAC nucleotide sequence comprising SEQ ID NO: 1; and (b) anucleic acid encoding a modified T cell receptor (TCR) having anaffinity for NY-ESO-1 on a target cell.